Capped pyrazinoylguanidine sodium channel blockers

ABSTRACT

The present invention relates to sodium channel blockers. The present invention also includes a variety of methods of treatment using these inventive sodium channel blockers.

CONTINUING APPLICATION DATA

This application claims benefit of U.S. Provisional Application Ser. No.60/495,725, filed on Aug. 18, 2003, and incorporated herein by referencein its entirety

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to sodium channel blockers. The presentinvention also includes a variety of methods of treatment using theseinventive sodium channel blockers.

2. Description of the Background

The mucosal surfaces at the interface between the environment and thebody have evolved a number of “innate defense”, i.e., protectivemechanisms. A principal form of such innate defense is to cleanse thesesurfaces with liquid. Typically, the quantity of the liquid layer on amucosal surface reflects the balance between epithelial liquidsecretion, often reflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupledwith water (and a cation counter-ion), and epithelial liquid absorption,often reflecting Na⁺ absorption, coupled with water and counter anion(Cl⁻ and/or HCO₃ ⁻). Many diseases of mucosal surfaces are caused by toolittle protective liquid on those mucosal surfaces created by animbalance between secretion (too little) and absorption (relatively toomuch). The defective salt transport processes that characterize thesemucosal dysfunctions reside in the epithelial layer of the mucosalsurface.

One approach to replenish the protective liquid layer on mucosalsurfaces is to “re-balance” the system by blocking Na⁺ channel andliquid absorption. The epithelial protein that mediates therate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺channel (ENaC). ENaC is positioned on the apical surface of theepithelium, i.e. the mucosal surface-environmental interface. Therefore,to inhibit ENaC mediated Na⁺ and liquid absorption, an ENaC blocker ofthe amiloride class (which blocks from the extracellular domain of ENaC)must be delivered to the mucosal surface and, importantly, be maintainedat this site, to achieve therapeutic utility. The present inventiondescribes diseases characterized by too little liquid on mucosalsurfaces and “topical” sodium channel blockers designed to exhibit theincreased potency, reduced mucosal abosrption, and slow dissociation(“unbinding” or detachment) from ENaC required for therapy of thesediseases.

Chronic bronchitis (CB), including the most common lethal genetic formof chronic bronchitis, cystic fibrosis (CF), are diseases that reflectthe body's failure to clear mucus normally from the lungs, whichultimately produces chronic airways infection. In the normal lung, theprimary defense against chronic intrapulmonary airways infection(chronic bronchitis) is mediated by the continuous clearance of mucusfrom bronchial airway surfaces. This function in health effectivelyremoves from the lung potentially noxious toxins and pathogens. Recentdata indicate that the initiating problem, i.e., the “basic defect,” inboth CB and CF is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance between the amount ofliquid and mucin on airway surfaces. This “airway surface liquid” (ASL)is primarily composed of salt and water in proportions similar to plasma(i.e., isotonic). Mucin macromolecules organize into a well defined“mucus layer” which normally traps inhaled bacteria and is transportedout of the lung via the actions of cilia which beat in a watery, lowviscosity solution termed the “periciliary liquid” (PCL). In the diseasestate, there is an imbalance in the quantities of mucus as ASL on airwaysurfaces. This results in a relative reduction in ASL which leads tomucus concentration, reduction in the lubricant activity of the PCL, anda failure to clear mucus via ciliary activity to the mouth. Thereduction in mechanical clearance of mucus from the lung leads tochronic bacterial colonization of mucus adherent to airway surfaces. Itis the chronic retention of bacteria, the failure of local antimicrobialsubstances to kill mucus-entrapped bacteria on a chronic basis, and theconsequent chronic inflammatory responses of the body to this type ofsurface infection, that lead to the syndromes of CB and CF.

The current afflicted population in the U.S. is 12,000,000 patients withthe acquired (primarily from cigarette smoke exposure) form of chronicbronchitis and approximately 30,000 patients with the genetic form,cystic fibrosis. Approximately equal numbers of both populations arepresent in Europe. In Asia, there is little CF but the incidence of CBis high and, like the rest of the world, is increasing.

There is currently a large, unmet medical need for products thatspecifically treat CB and CF at the level of the basic defect that causethese diseases. The current therapies for chronic bronchitis and cysticfibrosis focus on treating the symptoms and/or the late effects of thesediseases. Thus, for chronic bronchitis, β-agonists, inhaled steroids,anti-cholinergic agents, and oral theophyllines and phosphodiesteraseinhibitors are all in development. However, none of these drugs treateffectively the fundamental problem of the failure to clear mucus fromthe lung. Similarly, in cystic fibrosis, the same spectrum ofpharmacologic agents is used. These strategies have been complemented bymore recent strategies designed to clear the CF lung of the DNA(“Pulmozyme”; Genentech) that has been deposited in the lung byneutrophils that have futilely attempted to kill the bacteria that growin adherent mucus masses and through the use of inhaled antibiotics(“TOBI”) designed to augment the lungs' own killing mechanisms to ridthe adherent mucus plaques of bacteria. A general principle of the bodyis that if the initiating lesion is not treated, in this case mucusretention/obstruction, bacterial infections became chronic andincreasingly refractory to antimicrobial therapy. Thus, a major unmettherapeutic need for both CB and CF lung diseases is an effective meansof re-hydrating airway mucus (i.e., restoring/expanding the volume ofthe ASL) and promoting its clearance, with bacteria, from the lung.

R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use ofpyrazinoylguanidine sodium channel blockers for hydrating mucosalsurfaces. These compounds, typified by the well-known diureticsamiloride, benzamil, and phenamil are effective. However, thesecompounds suffer from the significant disadvantage that they are (1)relatively impotent, which is important because the mass of drug thatcan be inhaled by the lung is limited; (2) rapidly absorbed, whichlimits the half-life of the drug on the mucosal surface; and (3) arefreely dissociable from ENaC. The sum of these disadvantages embodied inthese well known diurectics produces compounds with insufficient potencyand/or effective half-life on mucosal surfaces to have therapeuticbenefit for hydrating mucosal surfaces.

Clearly, what is needed are drugs that are more effective at restoringthe clearance of mucus from the lungs of patients with CB/CF. The valueof these new therapies will be reflected in improvements in the qualityand duration of life for both the CF and the CB populations.

Other mucosal surfaces in and on the body exhibit subtle differences inthe normal physiology of the protective surface liquids on theirsurfaces but the pathophysiology of disease reflects a common theme,i.e., too little protective surface liquid. For example, in xerostomia(dry mouth) the oral cavity is depleted of liquid due to a failure ofthe parotid sublingual and submandibular glands to secrete liquiddespite continued Na⁺ (ENaC) transport mediated liquid absorption fromthe oral cavity. Similarly, keratoconjunctivitis sira (dry eye) iscaused by failure of lacrimal glands to secrete liquid in the face ofcontinued Na⁺ dependent liquid absorption on conjunctional surfaces. Inrhinosinusitis, there is an imbalance, as in CB, between mucin secretionand relative ASL depletion. Finally, in the gastrointestinal tract,failure to secrete Cl— (and liquid) in the proximal small intestine,combined with increased Na⁺ (and liquid) absorption in the terminalileum leads to the distal intestinal obstruction syndrome (DIOS). Inolder patients excessive Na⁺ (and volume) absorption in the descendingcolon produces constipation and diverticulitis.

Fifty million Americans and hundreds of millions of others around theworld suffer from high blood pressure and the subsequent sequale leadingto congestive heart failure and increasing mortality. It is the WesternWorld's leading killer and there is a need there for new medicines totreat these diseases. Thus, in addition, some of the novel sodiumchannel blockers of this invention can be designed to target the kidneyand as such they may be used as diuretics for the treatment ofhypertension, congestive heart failure (CHF) and other cardiovasculardiseases. These new agents may be used alone or in combination withbeta-blockers, ACE inhibitors, HMGCoA reductase inhibitors, calciumchannel blockers and other cardiovascular agents.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compounds that aremore potent and/or absorbed less rapidly from mucosal surfaces, and/orare less reversible as compared to known compounds.

It is another aspect of the present invention to provide compounds thatare more potent and/or absorbed less rapidly and/or exhibit lessreversibility, as compared to compounds such as amilorde, benzamil, andphenamil. Therefore, the compounds will give a prolonged pharmacodynamichalf-life on mucosal surfaces as compared to known compounds.

It is another object of the present invention to provide compounds whichare (1) absorbed less rapidly from mucosal surfaces, especially airwaysurfaces, as compared to known compounds and; (2) when absorbed frommusosal surfaces after administration to the mucosal surfaces, areconverted in vivo into metabolic derivitives thereof which have reducedefficacy in blocking sodium channels as compared to the administeredparent compound. It is another object of the present invention toprovide compounds that are more potent and/or absorbed less rapidlyand/or exhibit less reversibility, as compared to compounds such asamiloride, benzamil, and phenamil. Therefore, such compounds will give aprolonged pharmacodynamic half-life on mucosal surfaces as compared toprevious compounds.

It is another object of the present invention to provide compounds thattarget the kidney for use in the treatment of cardiovascular disease.

It is another object of the present invention to provide methods oftreatment that take advantage of the pharmacological properties of thecompounds described above.

In particular, it is an object of the present invention to providemethods of treatment which rely on rehydration of mucosal surfaces.

In particular, it is an object of the present invention to providemethods of treating cardiovascular disease.

The objects of the present invention may be accomplished with a class ofpyrazinoylguanidine compounds represented by formula (I):

wherein

X is hydrogen, halogen, trifluoromethyl, lower alkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl;

Y is hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, unsubstituted or substituted mononuclear aryl, or—N(R²)₂;

R¹ is hydrogen or lower alkyl;

each R² is, independently, —R⁷, —(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)-R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

R³ and R⁴ are each, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

wherein

-   -   each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸,        —O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,        —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,        —O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,        —O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,        —O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,        —O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,        —O(CH₂)_(m)-(Z)_(g)-R⁷,        —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,        —O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,        —(CH₂)_(n)—CO₂R⁷, —O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide,        —O-glucose,

each o is, independently, an integer from 0 to 10;

each p is an integer from 0 to 10;

with the proviso that the sum of o and p in each contiguous chain isfrom 1 to 10;

each x is, independently, O, NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, orrepresents a single bond;

wherein each R⁵ is, independently, Link —(CH₂)_(n)—CAP, Link—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, Link —(CH₂CH₂O)_(m)—CH₂-CAP, Link—(CH₂CH₂O)_(m)—CH₂CH₂-CAP, Link —(CH₂)_(n)-(Z)_(g)-CAP, Link—(CH₂)_(n)(Z)_(g)-(CH₂)_(m)—CAP, Link—(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP, Link—(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³-(Z)_(g)-CAP, Link—(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³-(Z)_(g)-CAP, Link—(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)-CAP, Link —NH—C(═O)—NH—(CH₂)_(m)-CAP, Link—(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)—C(═O)NR¹⁰R¹⁰, Link—(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)-CAP, Link —(CH₂)_(m)—C(═O)NR¹¹R¹¹, Link—(CH₂)_(m)—C(═O)NR¹²R¹², Link —(CH₂)_(n)-(Z)_(g)-(CH₂)_(m)-(Z)_(g)-CAP,Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)-CAP.

each Link is, independently, —O—, —(CH₂)_(n)—, —O(CH₂)_(m)—,—NR¹³—C(═O)—NR¹³, —NR¹³—C(═O)—(CH₂)_(m)—, —C(═O)NR¹³—(CH₂)_(m),—(CH₂)_(n)-Z_(g)-(CH₂)_(n), —S—, —SO—, —SO₂—, —SO₂NR⁷—, —SO₂NR¹⁰, or-Het-;

each CAP is, independently, thiazolidinedione, oxazolidinedione,heteroaryl-C(═O)N R¹³R¹³, heteroaryl-W, —CN, —O—C(═S)NR¹³R¹³, -Z_(g)R¹³,—CR¹⁰(Z_(g)R¹³)(Z_(g)R¹³), —C(═O)OAr, —C(═O)NR¹³Ar, imidazoline,tetrazole, tetrazole amide, —SO₂NHR¹³, —SO₂NH—C(R¹³R¹³)-(Z)_(g)R¹³, acyclic sugar or oligosaccharide, a cyclic amino sugar oroligosaccharide,

each Ar is, independently, phenyl, substituted phenyl, wherein thesubstituents of the substituted phenyl are 1-3 substituentsindependently selected from the group consisting of OH, OCH₃, NR¹³R¹³,Cl, F, and CH₃, or heteroaryl,

each W is independently, thiazolidinedione, oxazolidinedione,heteroaryl-C(═O)NR¹³R¹³, —CN, —O—C(═S)NR¹³R¹³, -Z_(g)R¹³,—CR¹⁰(Z_(g)R¹³)(Z_(g)R¹³), —C(═O)OAr, —C(═O)NR¹³Ar, imidazoline,tetrazole, tetrazole amide, —SO₂NHR¹³, —SO₂NH—C(R¹³R¹³)-(Z)_(g)-R¹³, acyclic sugar or oligosaccharide, a cyclic amino sugar oroligosaccharide,

each R⁶ is, independently, —R⁷, —OR⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

where when two R⁶ are —OR¹¹ and are located adjacent to each other on aphenyl ring, the alkyl moieties of the two R⁶ may be bonded together toform a methylenedioxy group; with the proviso that when at least two—CH₂OR⁸ are located adjacent to each other, the R⁸ groups may be joinedto form a cyclic mono- or di-substituted 1,3-dioxane or 1,3-dioxolane,

each R⁷ is, independently, hydrogen lower alkyl, phenyl, substitutedphenyl or —CH₂(CHOR)⁸ _(m)—R¹⁰;

each R⁸ is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹,glucuronide, 2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R¹³, —CON(R¹³)₂, —SO₂CH₂R¹³, or—C(═O)R¹³;

each R¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R¹³, —C(═O)NR¹³R¹³,—C(═O)R¹³, or (CH₂)_(m)—(CHOH)_(n)—CH₂OH;

each Z is, independently, CHOH, C(═O), —(CH₂)_(n)—, CHNR¹³R¹³, C═NR¹³,or NR¹³;

each R¹¹ is, independently, lower alkyl;

each R¹² is independently, —SO₂CH₃, —CO₂R¹³, —C(═O)NR¹³R¹³, —C(═O)R¹³,or —CH₂—(CHOH)_(n)—CH₂OH;

each R¹³ is, independently, hydrogen, R⁷, R¹⁰,

with the proviso that NR¹³R¹³ can be joined on itself to form a ringcomprising one of the following:

each Het is independently, —NR¹³, —S—, —SO—, or —SO₂—; —O—, —SO₂NR¹³—,—NHSO₂—, —NR¹³CO—, or —CONR¹³—;

each g is, independently, an integer from 1 to 6;

each m is, independently, an integer from 1 to 7;

each n is, independently, an integer from 0 to 7;

each Q is, independently, C—R⁵, C—R⁶, or a nitrogen atom, wherein atmost three Q in a ring are nitrogen atoms;

each V is, independently,

with the proviso that when V is attached directly to a nitrogen atom,then V can also be, independently, R⁷, R¹⁰, or (R¹¹)₂;

wherein for any of the above compounds when two —CH₂OR⁸ groups arelocated 1,2- or 1,3- with respect to each other the R⁸ groups may bejoined to form a cyclic mono- or di-substituted 1,3-dioxane or1,3-dioxolane;

wherein any of the above compounds can be a pharmaceutically acceptablesalt thereof, and wherein the above compounds are inclusive of allenantiomers, diastereomers, and racemic mixtures thereof.

The present invention also provides pharmaceutical compositions whichcontain a compound described above.

The present invention also provides a method of promoting hydration ofmucosal surfaces, comprising:

administering an effective amount of a compound represented by formula(I) to a mucosal surface of a subject.

The present invention also provides a method of restoring mucosaldefense, comprising:

topically administering an effective amount of compound represented byformula (I) to a mucosal surface of a subject in need thereof.

The present invention also provides a method of blocking ENaC,comprising:

contacting sodium channels with an effective amount of a compoundrepresented by formula (I).

The present invention also provides a method of promoting mucusclearance in mucosal surfaces, comprising:

administering an effective amount of a compound represented by formula(I) to a mucosal surface of a subject.

The present invention also provides a method of treating chronicbronchitis, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating cysticfibrosis, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating rhinosinusitis,comprising:

administering an effective amount of a compound represented by a formula(I) to a subject in need thereof.

The present invention also provides a method of treating nasaldehydration, comprising:

administering an effective amount of a compound represented by formula(I) to the nasal passages of a subject in need thereof.

In a specific embodiment, the nasal dehydration is brought on byadministering dry oxygen to the subject.

The present invention also provides a method of treating sinusitis,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating pneumonia,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of preventingventilator-induced pneumonia, comprising:

administering an effective compound represented by formula (I) to asubject by means of a ventilator.

The present invention also provides a method of treating asthma,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating primary ciliarydyskinesia, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating otitis media,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of inducing sputum fordiagnostic purposes, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating chronicobstructive pulmonary disease, comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating emphysema,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating dry eye,comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject in need thereof.

The present invention also provides a method of promoting ocularhydration, comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject.

The present invention also provides a method of promoting cornealhydration, comprising:

administering an effective amount of a compound represented by formula(I) to the eye of the subject.

The present invention also provides a method of treating Sjögren'sdisease, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating vaginaldryness, comprising:

administering an effective amount of a compound represented by formula(I) to the vaginal tract of a subject in need thereof.

The present invention also provides a method of treating dry skin,comprising:

administering an effective amount of a compound represented by formula(I) to the skin of a subject in need thereof.

The present invention also provides a method of treating dry mouth(xerostomia), comprising:

administering an effective amount of compound represented by formula (I)to the mouth of the subject in need thereof.

The present invention also provides a method of treating distalintestinal obstruction syndrome, comprising:

administering an effective amount of compound represented by formula (I)to a subject in need thereof.

The present invention also provides a method of treating esophagitis,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating constipation,comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof. In one embodiment of this method, thecompound is administered either orally or via a suppository or enema.

The present invention also provides a method of treating chronicdiverticulitis comprising:

administering an effective amount of a compound represented by formula(I) to a subject in need thereof.

The present invention also provides a method of treating hypertension,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of reducing blood pressure,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of treating edema,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting diuresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting natriuresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

The present invention also provides a method of promoting saluresis,comprising administering the compound represented by formula (I) to asubject in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the compounds offormula (I) are more potent and/or, absorbed less rapidly from mucosalsurfaces, especially airway surfaces, and/or less reversible frominteractions with ENaC as compared to compounds such as amiloride,benzamil, and phenamil. Therefore, the compounds of formula (I) have alonger half-life on mucosal surfaces as compared to these compounds.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) are converted in vivo into metabolicderivatives thereof that have reduced efficacy in blocking sodiumchannels as compared to the parent administered compound, after they areabsorbed from mucosal surfaces after administration. This importantproperty means that the compounds will have a lower tendency to causeundesired side-effects by blocking sodium channels located at untargetedlocations in the body of the recipient, e.g., in the kidneys.

The present invention is also based on the discovery that certaincompounds embraced by formula (I) target the kidney and thus may be usedas cardiovascular agents.

In the compounds represented by formula (I), X may be hydrogen, halogen,trifluoromethyl, lower alkyl, lower cycloalkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl. Halogen is preferred.

Examples of halogen include fluorine, chlorine, bromine, and iodine.Chlorine and bromine are the preferred halogens. Chlorine isparticularly preferred. This description is applicable to the term“halogen” as used throughout the present disclosure.

As used herein, the term “lower alkyl” means an alkyl group having lessthan 8 carbon atoms. This range includes all specific values of carbonatoms and subranges there between, such as 1,2, 3, 4, 5, 6, and 7 carbonatoms. The term “alkyl” embraces all types of such groups, e.g., linear,branched, and cyclic alkyl groups. This description is applicable to theterm “lower alkyl” as used throughout the present disclosure. Examplesof suitable lower alkyl groups include methyl, ethyl, propyl,cyclopropyl, butyl, isobutyl, etc.

Substituents for the phenyl group include halogens. Particularlypreferred halogen substituents are chlorine and bromine.

Y may be hydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio,halogen, lower alkyl, lower cycloalkyl, mononuclear aryl, or —N(R²)₂.The alkyl moiety of the lower alkoxy groups is the same as describedabove. Examples of mononuclear aryl include phenyl groups. The phenylgroup may be unsubstituted or substituted as described above. Thepreferred identity of Y is —N(R²)₂. Particularly preferred are suchcompounds where each R² is hydrogen.

R¹ may be hydrogen or lower alkyl. Hydrogen is preferred for R¹.

Each R² may be, independently, —R⁷, (CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)-R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n)—CO₂R⁷, or

Hydrogen and lower alkyl, particularly C₁-C₃ alkyl are preferred for R².Hydrogen is particularly preferred.

R³ and R⁴ may be, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, provided that at least one of R³ and R⁴ is a group represented byformula (A).

Preferred compounds are those where one of R³ and R⁴ is hydrogen and theother is represented by formula (A).

In formula (A), the moiety —(C(R^(L))₂)_(o)-x-(C(R^(L))₂)_(p)— definesan alkylene group bonded to the aromatic ring. The variables o and p mayeach be an integer from 0 to 10, subject to the proviso that the sum ofo and p in the chain is from 1 to 10. Thus, o and p may each be 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the sum of o and p is from 2 to6. In a particularly preferred embodiment, the sum of o and p is 4.

The linking group in the alkylene chain, x, may be, independently, O,NR¹⁰, C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or represents a single bond;

Therefore, when x represents a single bond, the alkylene chain bonded tothe ring is represented by the formula —(C(R^(L))₂)_(o+p)—, in which thesum o+p is from 1 to 10.

Each R^(L) may be, independently, —R⁷, —(CH₂)_(n)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

The preferred R^(L) groups include —H, —OH, —N(R⁷)₂, especially whereeach R⁷ is hydrogen.

In the alkylene chain in formula (A), it is preferred that when oneR^(L) group bonded to a carbon atoms is other than hydrogen, then theother R^(L) bonded to that carbon atom is hydrogen, i.e., the formula—CHR^(L). It is also preferred that at most two R^(L) groups in analkylene chain are other than hydrogen, where in the other R^(L) groupsin the chain are hydrogens. Even more preferably, only one R^(L) groupin an alkylene chain is other than hydrogen, where in the other R^(L)groups in the chain are hydrogens. In these embodiments, it ispreferable that x represents a single bond.

In another particular embodiment of the invention, all of the R^(L)groups in the alkylene chain are hydrogen. In these embodiments, thealkylene chain is represented by the formula

—(CH₂)_(o)-x-(CH₂)_(p)—.

Each R⁵ is, independently, Link-(CH₂)_(n)-CAP,Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, Link-(CH₂CH₂O)_(m)—CH₂-CAP,Link-(CH₂CH₂O)_(m)—CH₂CH₂-CAP, Link-(CH₂)_(n)-(Z)_(g)-CAP,Link-(CH₂)_(n)(Z)_(g)-(CH₂)_(m)-CAP,Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,Link-(CH₂)_(n)—(CHOR⁸)_(n)CH₂—NR¹³-(Z)_(g)-CAP,Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³-(Z)_(g)-CAP,Link-(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)-CAP, Link-NH—C(═O)—NH—(CH₂)_(m)-CAP,Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)—C(═O)NR¹⁰R¹⁰,Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)-CAP, Link-(CH₂)_(m)—C(═O)NR¹¹R¹¹,Link-(CH₂)_(m)—C(═O)NR¹²R¹²,Link-(CH₂)_(n)-(Z)_(g)-(CH₂)_(m)-(Z)_(g)-CAP,Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)-CAP.

Each Link is, independently, —O—, (CH₂)_(n)—, —O(CH₂)_(m)—,—NR¹³—C(═O)—NR¹³, —NR¹³—C(═O)—(CH₂)_(m)—, —C(═O)NR¹³—(CH₂)_(m),—(CH₂)_(n)-Z_(g)-(CH₂)_(n), —S—, —SO—, —SO₂—, SO₂NR⁷—, SO₂NR¹⁰—, or-Het-.

each CAP is, independently, thiazolidinedione, oxazolidinedione,heteroaryl-C(═O)NR¹³R¹³, heteroaryl-W, —CN, —O—C(═S)NR¹³R¹³, -Z_(g)R¹³,—CR¹⁰(Z_(g)R¹³)(Z_(g)R¹³), —C(═O)OAr, —C(═O)NR¹³Ar, imidazoline,tetrazole, tetrazole amide, —SO₂NHR¹³, —SO₂NH—C(R¹³R¹³)-(Z)_(g)-R¹³, acyclic sugar or oligosaccharide, a cyclic amino sugar oroligosaccharide,

Each Ar is, independently, phenyl, substituted phenyl, wherein thesubstituents of the substituted phenyl are 1-3 substituentsindependently selected from the group consisting of OH, OCH₃, NR¹³R¹³,Cl, F, and CH₃, or heteroaryl.

Each W is independently, thiazolidinedione, oxazolidinedione,heteroaryl-C(═O)NR¹³R¹³—CN, —O—C(═S)NR¹³R¹³, -Z_(g)R¹³,—CR¹⁰(Z_(g)R¹³)(Z_(g)R¹³), —C(═O)OAr, —C(═O)NR¹³Ar, imidazoline,tetrazole, tetrazole amide, —SO₂NHR¹³, —SO₂NH—C(R¹³R¹³)-(Z)_(g)-R¹³, acyclic sugar or oligosaccharide, a cyclic amino sugar oroligosaccharide,

Examples of heteroaryl include pyridyl, pyrazyl, tinazyl, furyl,furfuryl, thienyl, tetrazyl, thiazolidinedionyl and imidazoyl, pyrrolyl,furanyl, thiophenyl, quinolyl, indolyl, adenyl, pyrazolyl, thiazolyl,isoxazolyl, indolyl, benzimidazolyl, purinyl, quinolinyl, isoquinolinyl,pyridazyl, pyrimidyl, pyrazyl, 1,2,3-triazyl, 1,2,4-triazyl,1,3,5-triazyl, cinnolyl, phthalazyl, quinazolyl, quinoxalyl or pterdylgroups.

Each R⁶ is, independently, —R⁷, —OR⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

where when two R⁶ are —OR¹¹ and are located adjacent to each other on aphenyl ring, the alkyl moieties of the two R⁶ may be bonded together toform a methylenedioxy group;

with the proviso that when at least two —CH₂OR⁸ are located adjacent toeach other, the R⁸ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane.

In addition, one of more of the R⁶ groups can be one of the R⁵ groupswhich fall within the broad definition of R⁶ set forth above.

When two R⁶ are —OR¹¹ and are located adjacent to each other on a phenylring, the alkyl moieties of the two R⁶ groups may be bonded together toform a methylenedioxy group, i.e., a group of the formula —O—CH₂—O—.

As discussed above, R⁶ may be hydrogen. Therefore, 1, 2, 3, or 4 R⁶groups may be other than hydrogen. Preferably at most 3 of the R⁶ groupsare other than hydrogen.

Each g is, independently, an integer from 1 to 6. Therefore, each g maybe 1, 2, 3, 4, 5, or 6.

Each m is an integer from 1 to 7. Therefore, each m may be 1, 2, 3, 4,5, 6, or 7.

Each n is an integer from 0 to 7. Therefore, each n may be 0, 1, 2, 3,4, 5, 6, or 7.

Each Q in formula (A) is C—R⁵, C—R⁶, or a nitrogen atom, where at mostthree Q in a ring are nitrogen atoms. Thus, there may be 1, 2, or 3nitrogen atoms in a ring. Preferably, at most two Q are nitrogen atoms.More preferably, at most one Q is a nitrogen atom. In one particularembodiment, the nitrogen atom is at the 3-position of the ring. Inanother embodiment of the invention, each Q is either C—R⁵ or C—R⁶,i.e., there are no nitrogen atoms in the ring.

More specific examples of suitable groups represented by formula (A) areshown in formulas (B)-(E) below:

where o, x, p, R⁵, and R⁶, are as defined above;

where n is an integer from 1 to 10 and R⁵ is as defined above;

where n is an integer from 1 from 10 and R⁵ is as defined above;

where o, x, p, and R⁵ are as defined above.

In a preferred embodiment of the invention, Y is —NH₂.

In another preferred embodiment, R² is hydrogen.

In another preferred embodiment, R¹ is hydrogen.

In another preferred embodiment, X is chlorine.

In another preferred embodiment, R³ is hydrogen.

In another preferred embodiment, R^(L) is hydrogen.

In another preferred embodiment, o is 4.

In another preferred embodiment, p is 0.

In another preferred embodiment, the sum of o and p is 4.

In another preferred embodiment, x represents a single bond.

In another preferred embodiment, R⁶ is hydrogen.

In another preferred embodiment, at most one Q is a nitrogen atom.

In another preferred embodiment, no Q is a nitrogen atom.

In a preferred embodiment of the present invention:

X is halogen;

Y is —N(R⁷)₂;

R¹ is hydrogen or C₁-C₃ alkyl;

R² is —R⁷, —OR⁷, CH₂OR⁷, or —CO₂R⁷;

R³ is a group represented by formula (A); and

R⁴ is hydrogen, a group represented by formula (A), or lower alkyl;

In another preferred embodiment of the present invention:

X is chloro or bromo;

Y is —N(R⁷)₂;

R² is hydrogen or C₁-C₃ alkyl;

at most three R⁶ are other than hydrogen as described above;

at most three R^(L) are other than hydrogen as described above; and

at most 2 Q are nitrogen atoms.

In another preferred embodiment of the present invention:

Y is —NH₂;

In another preferred embodiment of the present invention:

R⁴ is hydrogen;

at most one R^(L) is other than hydrogen as described above;

at most two R⁶ are other than hydrogen as described above; and

at most 1 Q is a nitrogen atom.

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

In another preferred embodiment of the present invention the compound offormula (1) is represented by the formula:

The compounds of formula (I) may be prepared and used as the free base.Alternatively, the compounds may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example, chlorine, bromine, and iodine.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures of compounds within the scope of formula (I) are embraced bythe present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

Without being limited to any particular theory, it is believed that thecompounds of formula (I) function in vivo as sodium channel blockers. Byblocking epithelial sodium channels present in mucosal surfaces thecompounds of formula (I) reduce the absorption of water by the mucosalsurfaces. This effect increases the volume of protective liquids onmucosal surfaces, rebalances the system, and thus treats disease.

The present invention also provides methods of treatment that takeadvantage of the properties of the compounds of formula (I) discussedabove. Thus, subjects that may be treated by the methods of the presentinvention include, but are not limited to, patients afflicted withcystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronicobstructive airway disease, artificially ventilated patients, patientswith acute pneumonia, etc. The present invention may be used to obtain asputum sample from a patient by administering the active compounds to atleast one lung of a patient, and then inducing or collecting a sputumsample from that patient. Typically, the invention will be administeredto respiratory mucosal surfaces via aerosol (liquid or dry powders) orlavage.

Subjects that may be treated by the method of the present invention alsoinclude patients being administered supplemental oxygen nasally (aregimen that tends to dry the airway surfaces); patients afflicted withan allergic disease or response (e.g., an allergic response to pollen,dust, animal hair or particles, insects or insect particles, etc.) thataffects nasal airway surfaces; patients afflicted with a bacterialinfection e.g., staphylococcus infections such as Staphylococcus aureusinfections, Hemophilus influenza infections, Streptococcus pneumoniaeinfections, Pseudomonas aeuriginosa infections, etc.) of the nasalairway surfaces; patients afflicted with an inflammatory disease thataffects nasal airway surfaces; or patients afflicted with sinusitis(wherein the active agent or agents are administered to promote drainageof congested mucous secretions in the sinuses by administering an amounteffective to promote drainage of congested fluid in the sinuses), orcombined, Rhinosinusitis. The invention may be administered torhino-sinal surfaces by topical delivery, including aerosols and drops.

The present invention may be used to hydrate mucosal surfaces other thanairway surfaces. Such other mucosal surfaces include gastrointestinalsurfaces, oral surfaces, genito-urethral surfaces, ocular surfaces orsurfaces of the eye, the inner ear and the middle ear. For example, theactive compounds of the present invention may be administered by anysuitable means, including locally/topically, orally, or rectally, in aneffective amount.

The compounds of the present invention are also useful for treating avariety of functions relating to the cardiovascular system. Thus, thecompounds of the present invention are useful for use asantihypertensive agents. The compounds may also be used to reduce bloodpressure and to treat edema. In addition, the compounds of the presentinvention are also useful for promoting diuresis, natriuresis, andsaluresis. The compounds may be used alone or in combination with betablockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channelblockers and other cardiovascular agents to treat hypertension,congestive heart failure and reduce cardiovascular mortality.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

As discussed above, the compounds used to prepare the compositions ofthe present invention may be in the form of a pharmaceuticallyacceptable free base. Because the free base of the compound is generallyless soluble in aqueous solutions than the salt, free base compositionsare employed to provide more sustained release of active agent to thelungs. An active agent present in the lungs in particulate form whichhas not dissolved into solution is not available to induce aphysiological response, but serves as a depot of bioavailable drug whichgradually dissolves into solution.

Another aspect of the present invention is a pharmaceutical composition,comprising a compound of formula (I) in a pharmaceutically acceptablecarrier (e.g., an aqueous carrier solution). In general, the compound offormula (I) is included in the composition in an amount effective toinhibit the reabsorption of water by mucosal surfaces.

The compounds of the present invention may also be used in conjunctionwith a P2Y2 receptor agonist or a pharmaceutically acceptable saltthereof (also sometimes referred to as an “active agent” herein). Thecomposition may further comprise a P2Y2 receptor agonist or apharmaceutically acceptable salt thereof (also sometimes referred to asan “active agent” herein). The P2Y2 receptor agonist is typicallyincluded in an amount effective to stimulate chloride and watersecretion by airway surfaces, particularly nasal airway surfaces.Suitable P2Y2 receptor agonists are described in columns 9-10 of U.S.Pat. No. 6,264,975, U.S. Pat. No. 5,656,256, and U.S. Pat. No.5,292,498, each of which is incorporated herein by reference.

Bronchodiloators can also be used in combination with compounds of thepresent invention. These bronchodilators include, but are not limitedto, β-adrenergic agonists including but not limited to epinephrine,isoproterenol, fenoterol, albutereol, terbutalin, pirbuterol,bitolterol, metaproterenol, iosetharine, salmeterol xinafoate, as wellas anticholinergic agents including but not limited to ipratropiumbromide, as well as compounds such as theophylline and aminophylline.These compounds may be administered in accordance with known techniques,either prior to or concurrently with the active compounds describedherein. Additional procedures useful for the preparation are found inUSUSUS especially for the preparation of various

Another aspect of the present invention is a pharmaceutical formulation,comprising an active compound as described above in a pharmaceuticallyacceptable carrier (e.g., an aqueous carrier solution). In general, theactive compound is included in the composition in an amount effective totreat mucosal surfaces, such as inhibiting the reabsorption of water bymucosal surfaces, including airway and other surfaces.

The active compounds disclosed herein may be administered to mucosalsurfaces by any suitable means, including topically, orally, rectally,vaginally, ocularly and dermally, etc. For example, for the treatment ofconstipation, the active compounds may be administered orally orrectally to the gastrointestinal mucosal surface. The active compoundmay be combined with a pharmaceutically acceptable carrier in anysuitable form, such as sterile physiological or dilute saline or topicalsolution, as a droplet, tablet or the like for oral administration, as asuppository for rectal or genito-urethral administration, etc.Excipients may be included in the formulation to enhance the solubilityof the active compounds, as desired.

The active compounds disclosed herein may be administered to the airwaysurfaces of a patient by any suitable means, including as a spray, mist,or droplets of the active compounds in a pharmaceutically acceptablecarrier such as physiological or dilute saline solutions or distilledwater. For example, the active compounds may be prepared as formulationsand administered as described in U.S. Pat. No. 5,789,391 to Jacobus, thedisclosure of which is incorporated by reference herein in its entirety.

Solid or liquid particulate active agents prepared for practicing thepresent invention could, as noted above, include particles of respirableor non-respirable size; that is, for respirable particles, particles ofa size sufficiently small to pass through the mouth and larynx uponinhalation and into the bronchi and alveoli of the lungs, and fornon-respirable particles, particles sufficiently large to be retained inthe nasal airway passages rather than pass through the larynx and intothe bronchi and alveoli of the lungs. In general, particles ranging fromabout 1 to 5 microns in size (more particularly, less than about 4.7microns in size) are respirable. Particles of non-respirable size aregreater than about 5 microns in size, up to the size of visibledroplets. Thus, for nasal administration, a particle size in the rangeof 10-500 μm may be used to ensure retention in the nasal cavity.

In the manufacture of a formulation according to the invention, activeagents or the physiologically acceptable salts or free bases thereof aretypically admixed with, inter alia, an acceptable carrier. Of course,the carrier must be compatible with any other ingredients in theformulation and must not be deleterious to the patient. The carrier mustbe solid or liquid, or both, and is preferably formulated with thecompound as a unit-dose formulation, for example, a capsule, that maycontain 0.5% to 99% by weight of the active compound. One or more activecompounds may be incorporated in the formulations of the invention,which formulations may be prepared by any of the well-known techniquesof pharmacy consisting essentially of admixing the components.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate active agent composition may optionally contain adispersant which serves to facilitate the formulation of an aerosol. Asuitable dispersant is lactose, which may be blended with the activeagent in any suitable ratio (e.g., a 1 to 1 ratio by weight).

Active compounds disclosed herein may be administered to airway surfacesincluding the nasal passages, sinuses and lungs of a subject by asuitable means know in the art, such as by nose drops, mists, etc. Inone embodiment of the invention, the active compounds of the presentinvention and administered by transbronchoscopic lavage. In a preferredembodiment of the invention, the active compounds of the presentinvention are deposited on lung airway surfaces by administering anaerosol suspension of respirable particles comprised of the activecompound, which the subject inhales. The respirable particles may beliquid or solid. Numerous inhalers for administering aerosol particlesto the lungs of a subject are known.

Inhalers such as those developed by Inhale Therapeutic Systems, PaloAlto, Calif., USA, may be employed, including but not limited to thosedisclosed in U.S. Pat. Nos. 5,740,794; 5,654,007; 5,458,135; 5,775,320;and 5,785,049, each of which is incorporated herein by reference. TheApplicant specifically intends that the disclosures of all patentreferences cited herein be incorporated by reference herein in theirentirety. Inhalers such as those developed by Dura Pharmaceuticals,Inc., San Diego, Calif., USA, may also be employed, including but notlimited to those disclosed in U.S. Pat. Nos. 5,622,166; 5,577,497;5,645,051; and 5,492,112, each of which is incorporated herein byreference. Additionally, inhalers such as those developed by AradigmCorp., Hayward, Calif., USA, may be employed, including but not limitedto those disclosed in U.S. Pat. Nos. 5,826,570; 5,813,397; 5,819,726;and 5,655,516, each of which is incorporated herein by reference. Theseapparatuses are particularly suitable as dry particle inhalers.

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No.4,501,729, which is incorporated herein by reference. Nebulizers arecommercially available devices which transform solutions or suspensionsof the active ingredient into a therapeutic aerosol mist either by meansof acceleration of compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers consist of the active ingredient in aliquid carrier, the active ingredient comprising up to 40% w/w of theformulation, but preferably less than 20% w/w. The carrier is typicallywater (and most preferably sterile, pyrogen-free water) or diluteaqueous alcoholic solution. Perfluorocarbon carriers may also be used.Optional additives include preservatives if the formulation is not madesterile, for example, methyl hydroxybenzoate, antioxidants, flavoringagents, volatile oils, buffering agents and surfactants.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator.Aerosol generators for administering solid particulate medicaments to asubject produce particles which are respirable, as explained above, andgenerate a volume of aerosol containing predetermined metered dose ofmedicament at a rate suitable for human administration. One illustrativetype of solid particulate aerosol generator is an insufflator. Suitableformulations for administration by insufflation include finelycomminuted powders which may be delivered by means of an insufflator ortaken into the nasal cavity in the manner of a snuff. In theinsufflator, the powder (e.g., a metered dose thereof effective to carryout the treatments described herein) is contained in capsules orcartridges, typically made of gelatin or plastic, which are eitherpierced or opened in situ and the powder delivered by air drawn throughthe device upon inhalation or by means of a manually-operated pump. Thepowder employed in the insufflator consists either solely of the activeingredient or of powder blend comprising the active ingredient, asuitable powder diluent, such as lactose, and an optional surfactant.The active ingredient typically comprises of 0.1 to 100% w/w of theformulation. A second type of illustrative aerosol generator comprises ametered dose inhaler. Metered dose inhalers are pressurized aerosoldispensers, typically containing a suspension or solution formulation ofactive ingredient in a liquified propellant. During use, these devicesdischarge the formulation through a valve adapted to deliver a meteredvolume, typically from 10 to 150 μl, to produce a fine particle spraycontaining the active ingredient. Suitable propellants include certainchlorofluorocarbon compounds, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof.The formulation may additionally contain one of more co-solvents, forexample, ethanol, surfactants, such as oleic acid or sorbitan trioleate,antioxidants and suitable flavoring agents.

The aerosol, whether formed from solid or liquid particles, may beproduced by the aerosol generator at a rate of from about 10 to 150liters per minute, more preferable from 30 to 150 liters per minute, andmost preferably about 60 liters per minute. Aerosols containing greateramounts of medicament may be administered more rapidly.

The dosage of the active compounds disclosed herein will vary dependingon the condition being treated and the state of the subject, butgenerally may be from about 0.01, 0.03, 0.05, 0.1 to 1, 5, 10 or 20 mgof the pharmaceutic agent, deposited on the airway surfaces. The dailydose may be divided among one or multiple unit dose administrations. Thegoal is to achieve a concentration of the pharmaceutic agents on lungairway surfaces of between 10⁻⁹-10⁴ M.

In another embodiment, they are administered by administering an aerosolsuspension of respirable or non-respirable particles (preferablynon-respirable particles) comprised of active compound, which thesubject inhales through the nose. The respirable or non-respirableparticles may be liquid or solid. The quantity of active agent includedmay be an amount of sufficient to achieve dissolved concentrations ofactive agent on the airway surfaces of the subject of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻³, 10⁻², 10⁻¹ moles/liter, and more preferablyfrom about 10⁻⁹ to about 10⁻⁴ moles/liter.

The dosage of active compound will vary depending on the condition beingtreated and the state of the subject, but generally may be an amountsufficient to achieve dissolved concentrations of active compound on thenasal airway surfaces of the subject from about 10⁻⁹, 10⁻⁸, 10⁻⁷ toabout 10⁻³, 10⁻², or 10⁻¹ moles/liter, and more preferably from about10⁻⁷ to about 10⁻⁴ moles/liter. Depending upon the solubility of theparticular formulation of active compound administered, the daily dosemay be divided among one or several unit dose administrations. The dailydose by weight may range from about 0.01, 0.03, 0.1, 0.5 or 1.0 to 10 or20 milligrams of active agent particles for a human subject, dependingupon the age and condition of the subject. A currently preferred unitdose is about 0.5 milligrams of active agent given at a regimen of 2-10administrations per day. The dosage may be provided as a prepackagedunit by any suitable means (e.g., encapsulating a gelatin capsule).

In one embodiment of the invention, the particulate active agentcomposition may contain both a free base of active agent and apharmaceutically acceptable salt to provide both early release andsustained release of active agent for dissolution into the mucussecretions of the nose. Such a composition serves to provide both earlyrelief to the patient, and sustained relief over time. Sustained relief,by decreasing the number of daily administrations required, is expectedto increase patient compliance with the course of active agenttreatments.

Pharmaceutical formulations suitable for airway administration includeformulations of solutions, emulsions, suspensions and extracts. Seegenerally, J. Naim, Solutions, Emulsions, Suspensions and Extracts, inRemington: The Science and Practice of Pharmacy, chap. 86 (19^(th) ed.1995), incorporated herein by reference. Pharmaceutical formulationssuitable for nasal administration may be prepared as described in U.S.Pat. Nos. 4,389,393 to Schor; 5,707,644 to Illum; 4,294,829 to Suzuki;and 4,835,142 to Suzuki, the disclosures of which are incorporated byreference herein in their entirety.

Mists or aerosols of liquid particles comprising the active compound maybe produced by any suitable means, such as by a simple nasal spray withthe active agent in an aqueous pharmaceutically acceptable carrier, suchas a sterile saline solution or sterile water. Administration may bewith a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. Seee.g. U.S. Pat. Nos. 4,501,729 and 5,656,256, both of which areincorporated herein by reference. Suitable formulation is for use in anasal droplet or spray bottle or in nebulizers consist of the activeingredient in a liquid carrier, the active ingredient comprising up to40% w/w of the formulation, but preferably less than 20% w/w. Typicallythe carrier is water (and most preferably sterile, pyrogen-free water)or dilute aqueous alcoholic solution, preferably made in a 0.12% to 0.8%solution of sodium chloride. Optional additives include preservatives ifthe formulation is not made sterile, for example, methylhydroxybenzoate, antioxidants, flavoring agents, volatile oils,buffering agents, osmotically active agents (e.g. mannitol, xylitol,erythritol) and surfactants.

Compositions containing respirable or non-respirable dry particles ofmicronized active agent may be prepared by grinding the dry active agentwith a mortar and pestle, and then passing the micronized compositionthrough a 400 mesh screen to break up or separate out largeagglomerates.

The particulate composition may optionally contain a dispersant whichserves to facilitate the formation of an aerosol. A suitable dispersantis lactose, which may be blended with the active agent in any suitableratio (e.g., a 1 to 1 ratio by weight).

The compounds of formula (I) may be synthesized according to proceduresknown in the art. A representative synthetic procedure is shown in thescheme below:

These procedures are described in, for example, E. J. Cragoe, “TheSynthesis of Amiloride and Its Analogs” (Chapter 3) in Amiloride and ItsAnalogs, pp. 25-36, incorporated herein by reference. Other methods ofpreparing the compounds are described in, for example, U.S. Pat. No.3,313,813, incorporated herein by reference. See in particular MethodsA, B, C, and D described in U.S. Pat. No. 3,313,813. Other methodsuseful for the preparation of these compounds, especially for thepreparation of the novel HNR3R4 fragment are described in, for example,229929US, 233377US, and 234105US, incorporated herein by reference.Schemes 1 to 11 are representative, but limited to, of procedures usedto prepare the Sodium Channel Blockers described herein.

Several assays may be used to characterize the compounds of the presentinvention. Representative assays are discussed below.

In Vitro Measure of Sodium Channel Blocking Activity and Reversibility

One assay used to assess mechanism of action and/or potency of thecompounds of the present invention involves the determination of lumenaldrug inhibition of airway epithelial sodium currents measured undershort circuit current (I_(SC)) using airway epithelial monolayersmounted in Ussing chambers. Cells obtained from freshly excised human,dog, sheep or rodent airways are seeded onto porous 0.4 micron Snapwell™Inserts (CoStar), cultured at air-liquid interface (ALI) conditions inhormonally defined media, and assayed for sodium transport activity(I_(SC)) while bathed in Krebs Bicarbonate Ringer (KBR) in Usingchambers. All test drug additions are to the lumenal bath with half-logdose addition protocols (from 1×10⁻¹¹ M to 3×10⁻⁵ M), and the cumulativechange in ISc (inhibition) recorded. All drugs are prepared in dimethylsulfoxide as stock solutions at a concentration of 1×10⁻² M and storedat −20° C. Eight preparations are typically run in parallel; twopreparations per run incorporate amiloride and/or benzamil as positivecontrols. After the maximal concentration (5×10⁻⁵ M) is administered,the lumenal bath is exchanged three times with fresh drug-free KBRsolution, and the resultant I_(SC) measured after each wash forapproximately 5 minutes in duration. Reversibility is defined as thepercent return to the baseline value for sodium current after the thirdwash. All data from the voltage clamps are collected via a computerinterface and analyzed off-line.

Dose-effect relationships for all compounds are considered and analyzedby the Prism 3.0 program. IC₅₀ values, maximal effective concentrations,and reversibility are calculated and compared to amiloride and benzamilas positive controls.

Pharmacological Assays of Absorption (1) Apical Disappearance Assay

Bronchial cells (dog, human, sheep, or rodent cells) are seeded at adensity of 0.25×10⁶/cm² on a porous Transwell-Col collagen-coatedmembrane with a growth area of 1.13 cm² grown at an air-liquid interfacein hormonally defined media that promotes a polarized epithelium. From12 to 20 days after development of an air-liquid interface (ALI) thecultures are expected to be >90% ciliated, and mucins will accumulate onthe cells. To ensure the integrity of primary airway epithelial cellpreparations, the transepithelial resistance (R_(t)) and transepithelialpotential differences (PD), which are indicators of the integrity ofpolarized nature of the culture, are measured. Human cell systems arepreferred for studies of rates of absorption from apical surfaces. Thedisappearance assay is conducted under conditions that mimic the “thin”films in vivo (˜25 μl) and is initiated by adding experimental sodiumchannel blockers or positive controls (amiloride, benzamil, phenamil) tothe apical surface at an initial concentration of 10 μM. A series ofsamples (5 μl volume per sample) is collected at various time points,including 0, 5, 20, 40, 90 and 240 minutes. Concentrations aredetermined by measuring intrinsic fluorescence of each sodium channelblocker using a Fluorocount Microplate Fluorometer or HPLC. Quantitativeanalysis employs a standard curve generated from authentic referencestandard materials of known concentration and purity. Data analysis ofthe rate of disappearance is performed using nonlinear regression, onephase exponential decay (Prism V 3.0).

2. Confocal Microscopy Assay of Amiloride Congener Uptake

Virtually all amiloride-like molecules fluoresce in the ultravioletrange. This property of these molecules may be used to directly measurecellular update using x-z confocal microscopy. Equimolar concentrationsof experimental compounds and positive controls including amiloride andcompounds that demonstrate rapid uptake into the cellular compartment(benzamil and phenamil) are placed on the apical surface of airwaycultures on the stage of the confocal microscope. Serial x-z images areobtained with time and the magnitude of fluorescence accumulating in thecellular compartment is quantitated and plotted as a change influorescence versus time.

3. In Vitro Assays of Compound Metabolism

Airway epithelial cells have the capacity to metabolize drugs during theprocess of transepithelial absorption. Further, although less likely, itis possible that drugs can be metabolized on airway epithelial surfacesby specific ectoenzyme activities. Perhaps more likely as anecto-surface event, compounds may be metabolized by the infectedsecretions that occupy the airway lumens of patients with lung disease,e.g. cystic fibrosis. Thus, a series of assays is performed tocharacterize the compound metabolism that results from the interactionof test compounds with human airway epithelia and/or human airwayepithelial lumenal products.

In the first series of assays, the interaction of test compounds in KBRas an “ASL” stimulant are applied to the apical surface of human airwayepithelial cells grown in the T-Col insert system. For most compounds,metabolism (generation of new species) is tested for using highperformance liquid chromatography (HPLC) to resolve chemical species andthe endogenous fluorescence properties of these compounds to estimatethe relative quantities of test compound and novel metabolites. For atypical assay, a test solution (25 μl KBR, containing 10 μM testcompound) is placed on the epithelial lumenal surface. Sequential 5 to10 μl samples are obtained from the lumenal and serosal compartments forHPLC analysis of (1) the mass of test compound permeating from thelumenal to serosal bath and (2) the potential formation of metabolitesfrom the parent compound. In instances where the fluorescence propertiesof the test molecule are not adequate for such characterizations,radiolabeled compounds are used for these assays. From the HPLC data,the rate of disappearance and/or formation of novel metabolite compoundson the lumenal surface and the appearance of test compound and/or novelmetabolite in the basolateral solution is quantitated. The data relatingthe chromatographic mobility of potential novel metabolites withreference to the parent compound are also quantitated.

To analyze the potential metabolism of test compounds by CF sputum, a“representative” mixture of expectorated CF sputum obtained from 10 CFpatients (under IRB approval) has been collected. The sputum has been besolubilized in a 1:5 mixture of KBR solution with vigorous vortexing,following which the mixture was split into a “neat” sputum aliquot andan aliquot subjected to ultracentrifugation so that a “supernatant”aliquot was obtained (neat=cellular; supernatant=liquid phase). Typicalstudies of compound metabolism by CF sputum involve the addition ofknown masses of test compound to “neat” CF sputum and aliquots of CFsputum “supernatant” incubated at 37° C., followed by sequentialsampling of aliquots from each sputum type for characterization ofcompound stability/metabolism by HPLC analysis as described above. Asabove, analysis of compound disappearance, rates of formation of novelmetabolities, and HPLC mobilities of novel metabolites are thenperformed.

4. Pharmacological Effects and Mechanism of Action of the Drug inAnimals

The effect of compounds for enhancing mucociliary clearance (MCC) can bemeasured using an in vivo model described by Sabater et al., Journal ofApplied Physiology, 1999, pp. 2191-2196, incorporated herein byreference.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Preparation of Sodium Channel Blockers

Materials and methods. All reagents and solvents were purchased fromAldrich Chemical Corp. and used without further purification. NMRspectra were obtained on either a Bruker WM 360 (¹H NMR at 360 MHz and¹³C NMR at 90 MHz) or a Bruker AC 300 (¹H NMR at 300 MHz and ¹³C NMR at75 MHz). Flash chromatography was performed on a Flash Elute™ systemfrom Elution Solution (PO Box 5147, Charlottesville, Va. 22905) chargedwith a 90 g silica gel cartridge (40M FSO-0110-040155, 32-63 μm) at 20psi (N₂). GC-analysis was performed on a Shimadzu GC-17 equipped with aHeliflex Capillary Column (Alltech); Phase: AT-1, Length: 10 meters, ID:0.53 mm, Film: 0.25 micrometers. GC Parameters: Injector at 320° C.,Detector at 320° C., FID gas flow: H₂ at 40 ml/min, Air at 400 ml/min.Carrier gas: Split Ratio 16:1, N₂ flow at 15 ml/min, N₂ velocity at 18cm/sec. The temperature program is 70° C. for 0-3 min, 70-300° C. from3-10 min, 300° C. from 10-1 15 min.

HPLC analysis was performed on a Gilson 322 Pump, detector U/Vis-156 at360 nm, equipped with a Microsorb MV C8 column, 100 A, 25 cm. Mobilephase: A=acetonitrile with 0.1% TFA, B=water with 0.1% TFA. Gradientprogram: 95:5 B:A for 1 min, then to 20:80 B:A over 7 min, then to 100%A over 1 min, followed by washout with 100% A for 11 min, flow rate: 1ml/min.

Example 1 Synthesis ofN-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-(4-{4-[3-(1H-tetrazol-5-yl)propoxy]phenyl}butyl)guanidinehydrochloride (PSA 17926)

{4-[4-(3-Cyanopropoxy)phenyl]butyl}carbamic acid benzyl ester (2)

A mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid benzyl ester 1(2.00 g, 6.70 mmol), 4-bromobutyronitrile (0.70 mL, 6.70 mmol), andpotassium carbonate (1.00 g, 7.4 mmol) in DMF (10 mL), was stirred at65° C. for 16 h. Solvent was removed by rotary evaporation and theresidue was taken up in ethyl acetate, washed with water and brine, andconcentrated under vacuum. The crude product was purified by flashsilica gel column chromatography eluting with ethyl acetate/CH₂Cl₂ (1:9,v/v) to give the desired product 2 as a white solid (1.80 g, 75% yield).¹H NMR (300 MHz, CDCl₃) δ 1.56 (m, 4H), 2.15 (m, 2H), 2.55 (m, 4H), 3.15(m, 2H), 4.00 (m, 2H), 4.70 (br s, 1H), 5.10 (s, 2H), 6.80 (d, 2H), 7.05(d, 2H), 7.30 (m, 5H). m/z (ESI): 367 [C₂₂H₂₆N₂O₃+H]⁺.

(4-{4-[3-(1H-Tetrazol-5-yl)propoxy]phenyl}butyl)carbamic acid benzylester (3)

A mixture of {4-[4-(3-cyanopropoxy)phenyl]butyl}carbamic acid benzylester 2 (0.90 g, 2.5 mmol), sodium azide (0.50 g, 7.5 mmol), andammonium chloride (0.40 g, 7.5 mmol) in DMF (7 mL), was stirred at 120°C. for 16 h. Inorganics were removed by vacuum filtration. The filtratewas diluted with ethyl acetate, and washed with water and brine. Theorganic solution was dried over Na₂SO₄, filtered and concentrated. Theresidue was taken up in ethyl acetate (5 mL) and diluted with hexanes(10 mL). Solid precipitates were collected by suction filtration andpurified by flash silica gel column chromatography eluting withmethanol/dichloromethane (1:50, v/v) to give the desired product 3 as awhite solid (0.78 g, 76% yield). ¹H NMR (300 MHz, CD₃OD) δ 1.51 (m, 4H),2.20 (m, 2H), 2.50 (m, 2H), 3.10 (m, 4H), 4.00 (m, 2H), 5.00 (s, 2H),6.75 (d, 2H), 7.05 (d, 2H), 7.30 (m, 5H). m/z (ESI): 410[C₂₂H₂₇N₅O₃+H]⁺.

4-{4-[3-(1H-Tetrazol-5-yl)propoxy]phenyl}butylamine (4)

A solution of (4-{4-[3-(1H-tetrazol-5-yl)propoxy]phenyl}butyl)carbamicacid benzyl ester 3 (0.30 g, 0.73 mmol) in methanol (20 mL) anddichloromethane (5 mL) was stirred at room temperature overnight underhydrogen atmosphere in the presence of 10% palladium-on-carbon catalyst(0.1 g, 50% wet). The catalyst was removed by suction filtration, andthe filtrate was concentrated in vacuo to give the desired product 4 asa white solid (200 mg, 99% yield) which was used for the next stepwithout further purification. m/z (ESI): 276 [C₁₄H₂₁N₅O+H]⁺.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(4-{4-[3-(1H-tetrazol-5-yl)-propoxy]phenyl}butyl)guanidinehydrochloride (5, PSA 17926)

A solution of 4-{4-[3-(1H-tetrazol-5-yl)propoxy]phenyl}butylamine 4 (100mg, 0.36 mmol) and triethylamine (0.15 mL, 0.39 mmol) in absoluteethanol (2 mL) was stirred at 60° C. for 5 min, after which1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methyl-isothioureahydriodide (150 mg, 0.39 mol) was added in one portion. The reactionmixture was stirred at that temperature for 4 h and then cooled to roomtemperature. The reaction mixture was concentrated by rotaryevaporation. The crude residue was washed with water and filtered. Thefilter cake was further washed with dichloromethane. A dark yellow solid(140 mg, 80% yield) thus obtained was slurried in a mixture of methanoland dichloromethane (5/95, v/v). The solid was collected by suctionfiltration, and 40 mg of such solid was mixed with 3% aqueous HCl (4mL). The mixture was sonicated, stirred at room temperature for 15 minand filtered. The filter cake was dried under high vacuum to giveN-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-(4-{4-[3-(1H-tetrazol-5-yl)propoxy]phenyl}butyl)guanidinehydrochloride (5, PSA 17926) as a yellow solid. mp 125-127° C.(decomposed). ¹H NMR (300 MHz, CD₃OD) δ 1.70 (m, 4H), 2.22 (m, 2H), 2.60(m, 2H), 3.10 (m, 2H), 4.00 (m, 2H), 6.70 (d, 2H), 7.09 (d, 2H). m/z(ESI): 488 [C₂₀H₂₆ClN₁₁O₂+H]⁺.

Example 2 Synthesis of dimethylthiocarbamic acidO-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenyl)ester (PSA 17846)

2-[4-(4-Hydroxyphenyl)butyl]isoindole-1,3-dione (8)

A mixture of 4-(4-aminobutyl)phenol hydrobromide 6 (8.2 g, 33.5 mmol),phthalic anhydride 7 (5.0 g, 33.8 mmol), and triethylamine (4.6 mL, 33.5mmol) in chloroform (50 mL) was stirred at reflux for 18 h, cooled toroom temperature and concentrated by rotary evaporation. The residue wasdissolved in acetic acid (50 mL) and stirred at 100° C. for 3 h. Solventwas evaporated and the resulting residue was purified by flash silicagel column chromatography eluting with CH₂Cl₂/EtOAc/hexanes (8:1:1, v/v)to give the desired product 8 as a white powder (4.1 g, 41% yield). ¹HNMR (300 MHz, DMSO-d₆) δ 1.57 (m, 4H), 2.46 (m, 2H), 3.58 (m, 2H), 6.64(d, 2H), 6.95 (d, 2H), 7.82 (m, 4H), 9.12 (s, 1H). m/z (ESI): 296[C₁₈H₁₇NO₃+H]⁺.

Dimethylthiocarbamic acidO-{4-[4-(1,3-dioxo-1,3-dihydroisoindol-2-yl)butyl]-phenyl}ester (9)

A suspension of sodium hydride (60% in mineral oil, 0.44 g, 0.11 mmol)in anhydrous DMF (10 mL) was cooled to 0° C. and added to a solution of2-[4-(4-hydroxyphenyl)-butyl]isoindole-1,3-dione 8 (2.95 g, 10 mmol) inDMF (15 mL). The mixture was stirred at 0° C. for 30 min and then atroom temperature for an additional one hour. A solution ofdimethylthiocarbamic acid chloride (1.35 g, 11 mmol) in DMF (10 mL) wasthen added. The reaction mixture was stirred at room temperature firstfor 16 h and then at 50° C. for 1 h, cooled back to room temperature andquenched with methanol (10 mL). The mixture was concentrated undervacuum and the residue was purified by flash silica gel columnchromatography eluting with CH₂Cl₂/hexanes/EtOAc (10:1:0.2, v/v) to givethe desired product 9 as a yellowish solid (2.27 g, 59% yield). ¹H NMR(300 MHz, CDCl₃) δ 1.72 (m, 4H), 2.67 (m, 2H), 3.33 (s, 3H), 3.45 (s,3H), 3.71 (m, 2H), 6.95 (d, 2H), 7.18 (d, 2H), 7.70 (m, 2H), 7.84 (m,2H). m/z (ESI): 383 [C₂₁H₂₂N₂O₃S+H]⁺.

Dimethylthiocarbamic acid O-[4-(4-aminobutyl)phenyl]ester (10)

A mixture of dimethylthiocarbamic acidO-{4-[4-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-butyl]phenyl}ester 9 (0.30g, 0.80 mmol) and methylamine (2M in methanol, 10 mL, 20 mmol) wasstirred at room temperature overnight. Solvent was removed by rotaryevaporation and the residue was purified by flash silica gel columnchromatography (Biotage) eluting with chloroform/methanol/concentratedammonium hydroxide (10:1:0.1, v/v) to give dimethylthiocarbamic acidO-[4-(4-aminobutyl)phenyl]ester (10) as a clear colorless oil (118 mg,46% yield). ¹H NMR (300 MHz, CD₃OD) δ 1.70 (m, 4H), 2.70 (m, 4H), 3.34(s, 3H), 3.46 (s, 3H), 6.96 (d, 2H), 7.20 (d, 2H). m/z (ESI): 253[C₁₃H₂₀N₂OS+H]⁺.

Dimethylthiocarbamic acidO-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidinio]butyl}phenyl)ester (11, PSA 17846)

A solution of dimethylthiocarbamic acid O-[4-(4-aminobutyl)phenyl]ester10 (15 mg, 0.45 mmol), triethylamine (0.30 mL, 2.2 mmol), and1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (175 mg, 0.45 mmol) in anhydrous THF (6 mL) was stirred atreflux for 3 h and then cooled to room temperature. The reaction mixturewas concentrated by rotary evaporation. The crude residue was purifiedby flash silica gel column chromatography (Biotage) eluting withchloroform/methanol/concentrated ammonium hydroxide (15:1:0.1, v/v) togive the desired product 11 as a yellow solid (180 mg, 86% yield). mp102-105° C. ¹H NMR (300 MHz, CD₃OD) δ 1.70 (m, 4H), 2.65 (m, 2H), 3.20(m, 2H), 3.30 (s, 3H), 3.40 (s, 3H), 6.95 (d, 2H), 7.20 (d, 2H). m/z(ESI): 465 [C₁₉H₂₅ClN₈O₂S+H]⁺.

Example 3 Synthesis of(2S)-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}benzenesulfonylamino)-3-methylbutyramide(PSA 19008)

4-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)butyl]benzenesulfonyl chloride(13)

2-(4-Phenylbutyl)isoindole-1,3-dione 12 (1.9 g, 6.8 mmol) was added tochlorosulfonic acid (10 mL, 138 mmol) at 0° C. and the mixture wasstirred for 1 h at the temperature. After storing in refrigerator at −5°C. overnight, the reaction mixture was poured onto crushed ice (100 g)and precipitates were collected by a suction filtration and dried underhigh vacuum to afford the desired product 13 (2.48 g, 99% yield). ¹H NMR(300 MHz, CDCl₃) δ 1.70 (m, 4H), 2.78 (m, 2H), 3.70 (m, 2H), 7.40 (d,2H) 7.70 (d, 2H), 7.85 (d, 2H), 7.95 (d, 2H).

(2S)-{4-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)butyl]benzenesulfonylamino}-3-methylbutyramide(14)

4-[4-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)butyl]benzenesulfonyl chloride13 (0.45 g, 1.19 mmol) was dissolved in dry DMF (5 mL), and added to asolution of N-methylmorpholine (3 mL) and (2S)-amino-3-methylbutyramide(0.18 g, 1.19 mmol) in DMF (10 mL). The reaction mixture was stirred atroom temperature for 66 h. Solvent was removed by rotary evaporation andthe residue was purified by flash silica gel chromatography eluting withchloroform/methanol/concentrated ammonium hydroxide (15:1:0.1, v/v) togive the desired product 14 as a white powder (0.41 g, 73% yield). ¹HNMR (300 MHz, DMSO-d₆) δ 0.72 (d, 3H), 0.76 (d, 3H), 1.77 (m, 4H), 1.79(m, 1H), 2.68 (m, 2H), 3.40 (m, 1H), 3.60 (m, 2H), 6.92 (s, 1H), 7.21(s, 1H), 7.34 (d, 2H) 7.50 (d, 1H), 7.65 (d, 2H), 7.82 (m, 4H).

(2S)-[4-(4-Aminobutyl)benzenesulfonylamino]-3-methylbutyramide (15)

A mixture of(2S)-{4-[4-(1,3-dioxo-1,3-dihydroisoindol-2-yl)butyl]-benzenesulfonylamino}-3-methylbutyramide14 (0.40 g, 0.87 mmol) and methylamine (2 M in methanol, 20 mL, 40 mmol)was stirred at room temperature overnight. Solvent was removed by rotaryevaporation and the residue was purified by flash silica gel columnchromatography eluting with chloroform/methanol/concentrated ammoniumhydroxide (3:1:0.1, v/v) to give(2S)-[4-(4-aminobutyl)benzenesulfonylamino]-3-methyl-butyramide (15) asa white powder (156 mg, 54% yield). ¹H NMR (300 MHz, CD₃OD) δ 0.85 (d,3H), 0.87 (d, 3H), 1.66 (m, 4H), 1.90 (m, 1H), 2.69 (m, 4H), 3.51 (d,1H), 7.35 (d, 2H) 7.75 (d, 2H). m/z (ESI): 328 [C₁₅H₂₅N₃O₃S+H]⁺.

(2S)-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-benzenesulfonylamino)-3-methylbutyramide(16, PSA 19008)

A solution of(2S)-[4-(4-aminobutyl)benzenesulfonylamino]-3-methylbutyramide 15 (156mg, 0.47 mmol), diisopropylethylamine (0.60 mL, 3.0 mmol), and1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (230 mg, 0.61 mmol) in absolute ethanol (8 mL) was stirred at70° C. for 5 h and then cooled to room temperature. The reaction mixturewas concentrated by rotary evaporation. The crude residue was washedwith water, filtered and the crude solid product was purified by flashsilica gel column chromatography eluting withchloroform/methanol/concentrated ammonium hydroxide (5:1:0.1, v/v) togive the desired product as a yellow solid (137 mg, 54% yield). Part ofthe solid (86 mg) was further purified by semi-preparative HPLC(acetonitrile/water/0.1% TFA) to give the analytical pure sample whichwas then co-evaporated with 5% aqueous HCl to give the hydrochloridesalt 16. mp 154-156° C. (decomposed). ¹H NMR (300 MHz, CD₃OD) δ 0.85 (d,3H), 0.86 (d, 3H), 1.70 (m, 4H), 1.90 (m, 1H), 2.75 (m, 2H), 3.32 (m,2H), 3.52 (d, 1H), 7.35 (d, 2H), 7.75 (d, 2H). m/z (ESI): 540[C₂₁H₃₀ClN₉O₄S+H]⁺. [α]_(D) ²⁵+5.2° (c 0.50, MeOH).

Example 4 Synthesis of2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenoxy)-N-phenylacetamide(PSA 17482)

[4-(4-Phenylcarbamoylmethoxyphenyl)butyl]carbamic acid benzyl ester (18)

A mixture of [4-(4-benzyloxycarbonylaminobutyl)phenoxy]acetic acid (300mg, 0.84 mmol), aniline (0.15 mL, 1.70 mmol), DMAP (60 mg, 0.50 mmol)and EDC.HCl (320 mg, 1.70 mmol) in CH₂Cl₂ (30 mL) was stirred at roomtemperature for 66 h. The reaction mixture was concentrated under vacuumand the residue was subjected to flash silica gel column chromatographyeluting with methanol/CH₂Cl₂ (1:99, v/v) to give the desired amide 18 asa white solid (360 mg, 99% yield). ¹H NMR (300 MHz, CDCl₃) δ 1.55 (m,4H), 2.60 (m, 2H), 3.20 (m, 2H), 4.58 (s, 2H), 4.70 (br s, 1H), 5.10 (s,2H), 6.88 (d, 2H), 7.15 (m, 3H), 7.35 (m, 7H), 7.58 (d, 2H), 8.25 (s,1H). m/z (ESI): 433 [C₂₆H₂₈N₂O₄ ⁺H]⁺.

2-[4-(4-Aminobutyl)phenoxy]-N-phenylacetamide (19)

A solution of [4-(4-phenylcarbamoylmethoxyphenyl)butyl]carbamic acidbenzyl ester 18 (0.30 g, 0.69 mmol) in ethanol (10 mL), THF (6 mL), andacetic acid (2 mL) was stirred at room temperature for 2 h underhydrogen atmosphere in the presence of 10% Pd/C catalyst (0.2 g, 50%wet). The catalyst was removed by suction filtration and the filtratewas concentrated in vacuo. The residue was purified by flash silica gelcolumn chromatography eluting with CH₂Cl₂/methanol/concentrated ammoniumhydroxide (30:1:0, 30:1:0.3, v/v) to give the desired amine 19 as awhite solid (200 mg, 97% yield). ¹H NMR (300 MHz, CD₃OD) δ 1.60 (m, 4H),2.55 (m, 2H), 2.70 (m, 2H), 4.60 (s, 2H), 6.88 (d, 2H), 7.15 (m, 3H),7.35 (m, 2H), 7.58 (d, 2H), 8.25 (s, 1H). m/z (ESI): 299[C₁₈H₂₂N₂O₂+H]⁺.

2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-N-phenylacetamide(20, PSA 17482).

A solution of 2-[4-(4-aminobutyl)phenoxy]-N-phenylacetamide 19 (100 mg,0.35 mmol) and triethylamine (0.14 mL, 1.00 mmol) in absolute ethanol (2mL) was stirred at 60° C. for 30 min, after which1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methyl-isothioureahydriodide (140 mg, 0.37 mmol) was added in one portion. The reactionmixture was stirred at that temperature for 4 h, cooled to roomtemperature, and concentrated by rotary evaporation. The crude residuewas triturated with water and filtered. The filter cake was purified byflash silica gel column chromatography eluting withdichloromethane/methanol/concentrated ammonium hydroxide (500:10:0,500:10:1, 200:10:1, v/v) to give2-(4-{4-[N′-(3,5-diamino-6-chloro-pyrazine-2-carbonyl)guanidino]butyl}phenoxy)-N-phenylacetamide(20, PSA 17482) as a yellow solid (120 mg, 67% yield). mp 168-170° C. ¹HNMR (300 MHz, DMSO-d₆) δ 1.55 (m, 4H), 2.55 (m, 21), 3.16 (m, 21), 4.65(s, 2H), 6.60 (br s, 2H), 6.90 (d, 21), 7.08 (m, 2H), 7.15 (d, 2H), 7.30(m, 5H), 7.60 (d, 2H), 9.00 (br s, 1H), 10.00 (br s, 1H). m/z (ESI): 511[C₂₄H₂₇ClN₈O₃+H]⁺.

Example 5 Synthesis ofN-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-(4-{4-[3-(1H-imidazol-2-yl)propoxy]phenyl}butyl)guanidine(PSA 23022)

4-{4-[3-(1H-Imidazol-2-yl)propoxy]phenyl}butylamine (21)

Compound 2 (0.156 g, 0.425 mmol) was dissolved in anhydrous ethanol (10mL). To the solution was bubbled anhydrous HCl gas for 3 min. Thereaction vessel was sealed and the mixture was stirred at roomtemperature for 48 h, and then concentrated to dryness under vacuum. Theresulting residue was dissolved in anhydrous methanol (5 mL). To thenewly formed solution was added 2,2-dimethoxyethylamine (0.097 mL, 0.891mmol) in one portion. After stirring at room temperature overnight,temperature was raised to reflux which was maintained for another 3 hbefore the mixture was cooled to ambient temperature. Solvent wasremoved under vacuum and the residue was treated with 1.2 N HCl aqueoussolution at 80° C. for 2 hours. The mixture was then cooled to ambienttemperature again and neutralized to pH ˜9 with powder K₂CO₃. Water wascompletely removed under vacuum and the residue was dissolved inmethanol. The methanol solution was loaded onto silica gel, and theproduct was eluted with a mixture of concentrated ammoniumhydroxide/MeOH/CH₂Cl₂ (1.8:18:81.2, v/v), affording the product 21 (27mg, 23% overall yield) as an off-white solid. ¹H NMR (300 MHz, CD₃OD): δ1.60 (m, 4H), 2.14 (m, 2H), 2.56 (t, 2H), 2.76 (t, 2H), 2.86 (t, 2H),3.94 (t, 2H), 6.79 (d, 2H), 6.91 (s, 2H), 7.08 (d, 2H). m/z (APCI): 274[C₁₆H₂₃N₃O+H]⁺.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(4-{4-[3-(1H-imidazol-2-yl)propoxy]phenyl}butyl)guanidine(22, PSA 23022)

Compound 21 (23 mg, 0.084 mmol) was dissolved in a mixture of ethanol (3mL) and Hunig's base (0.074 mL, 0.421 mmol) at 65° C. over 15 min. Tothe solution was added1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (43 mg, 0.109 mmol) and the resulting mixture was stirred atthe above temperature for an additional 3 h before all liquid wasremoved under vacuum. The residue was chromatographed oil silica gel,eluting with a mixture of concentrated ammoniumhydroxide/methanol/dichloromethane (1.5:15:63.5, v/v), to afford thedesired product 22 (34 mg, 83% yield) as a yellow solid. mp 123-126° C.(decomposed), ¹H NMR (300 MHz, CD₃OD): δ 1.62 (m, 4H), 2.14 (m, 2H),2.58 (t, 2H), 2.88 (t, 2H), 3.21 (t, 2H), 3.94 (t, 2H), 6.77 (d, 2H),6.90 (s, 2H), 7.06 (d, 2H). m/z (APCI): 486 [C₂₂H₂₈ClN₉O₂+H]⁺.

Example 6 Synthesis of2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenoxy)-N,N-bis-(2-hydroxyethyl)acetamide(PSA 16826)

[4-(4-{[N,N-Bis-(2-hydroxyethyl)carbamoyl]methoxy}phenyl)butyl]carbamicacid benzyl ester (25)

A solution of [4-(4-benzyloxycarbonylaminobutyl)phenoxy]acetic acidethyl ester 23 (0.3 g, 0.78 mmol), 2-(2-hydroxyethylamino)ethanol 24(0.15 mL, 1.6 mmol), and ethanol (20 mL) was heated at 70° C. for 72hours. Solvent was evaporated in vacuo. The residue was purified byflash chromatography (silica gel, dichloromethane/methanol, 100:5, v/v)to provide[4-(4-{[N,N-bis-(2-hydroxyethyl)carbamoyl]methoxy}phenyl)-butyl]carbamicacid benzyl ester 25 [0.19 g, 100% based on the recovered startingmaterial (0.13 g)] as a pale yellow solid. ¹H NMR (300 MHz, CD₃OD) δ1.65 (m, 4H), 2.50 (m, 2H), 3.20 (m, 2H), 3.55 (m, 4H), 3.75 (m, 4H),4.80 (s, 2H), 5.10 (s, 2H), 6.85 (d, 2H), 7.10 (d, 2H), 7.40 (m, 5H).m/z (ESI): 445 [C₂₄H₃₂N₂O₆+H]⁺.

2-[4-(4-Aminobutyl)phenoxy]-N,N-bis-(2-hydroxyethyl)acetamide (26)

To a degassed solution of[4-(4-{[N,N-bis-(2-hydroxyethyl)carbamoyl]methoxy}phenyl)-butyl]carbamicacid benzyl ester 25 (0.19 g, 0.43 mmol) in ethanol (4 mL) was added 10%palladium on activated carbon (0.1 g, 50% wet). The mixture washydrogenated overnight at atmospheric hydrogen. The catalyst wasfiltered through a pad of diatomaceous earth and the solvent wasevaporated in vacuo. The residue was purified by flash chromatography(silica gel, 20-5:1:0.1-1 dichloromethane/methanol/concentrated ammoniumhydroxide, v/v) to provide 26 (0.09 g, 72%) as a colorless oil. ¹H NMR(300 MHz, CD₃OD) δ 1.56 (m, 4H), 2.56 (t, 2H), 2.65 (t, 1H), 3.29 (m,1H), 3.55 (m, 4H), 3.72 (m, 4H), 4.90 (s, 2H), 6.86 (d, 2H), 7.09 (d,2H). m/z (ESI): 311 [C₁₆H₂₆N₂O₄ ⁺H]⁺.

2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-N,N-bis-(2-hydroxyethyl)acetamide(27, PSA 16826)

1-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (0.13 g, 0.33 mmol) was added to a solution of2-[4-(4-aminobutyl)phenoxy]-N,N-bis-(2-hydroxyethyl)acetamide 26 (0.09g, 0.3 mmol), triethylamine (0.12 mL), and ethanol (1.7 mL). Thereaction mixture was stirred at 60° C. for 3 h. The solvent wasevaporated in vacuo. The residue was triturated with water and thenpurified by flash chromatography (silica gel, 20-10:1:0-0.2CH₂Cl₂/methanol/concentrated ammonium hydroxide, v/v) to provide2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)-N,N-bis-(2-hydroxyethyl)acetamide27 (0.1 g, 64%) as a yellow solid. mp 114-116° C. ¹H NMR (300 MHz,CD₃OD) δ 1.70 (m, 4H), 2.60 (m, 2H), 3.32 (m, 2H), 3.50 (m, 4H), 3.70(m, 4H), 4.81 (s, 2H), 6.85 (d, 2H), 7.10 (d, 2H). m/z (ESI): 523[C₂₂H₃₁ClN₈O₅+H]⁺.

Example 7 Synthesis of2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenoxy)-N,N-dimethylacetamidehydrochloride (PSA 16313)

[4-(4-Dimethylcarbamoylmethoxyphenyl)butyl]carbamic acid benzyl ester(28)

A mixture of [4-(4-benzyloxycarbonylaminobutyl)phenoxy]acetic acid ethylester 23 (0.50 g, 1.3 mmol) and dimethylamine (2.0 M in THF, 10 mL, 20mmol) in a sealed tube was heated at 55° C. for 48 h. The solvent wasevaporated in vacuo. The residue was purified by flash chromatography(silica gel, ethyl acetate/CH₂Cl₂, 1:4, 1:3, v/v) to provide[4-(4-dimethylcarbamoylmethoxyphenyl)butyl]carbamic acid benzyl ester 28(0.26 g, 52% yield) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.55 (m,4H), 2.55 (m, 2H), 2.90 (s, 3H), 3.05 (s, 3H), 3.20 (m, 2H), 4.65 (s,2H), 5.08 (s, 2H), 6.80 (d, 2H), 7.05 (d, 2H), 7.35 (m, 5H).

2-[4-(4-Aminobutyl)phenoxy]-N,N-dimethylacetamide (29)

To a degassed solution of[4-(4-dimethylcarbamoylmethoxyphenyl)butyl]carbamic acid benzyl ester(28) (0.26 g, 0.68 mmol) in ethanol (10 mL) was added 10% palladium onactivated carbon (0.1 g, 50% wet). The mixture was stirred at roomtemperature overnight under atmospheric hydrogen. The catalyst wasfiltered through a pad of diatomaceous earth and the solvent wasevaporated in vacuo. The residue was purified by flash chromatography(silica gel, dichloromethane/methanol/concentrated ammonium hydroxide,100:5:1, v/v) to provide2-[4-(4-aminobutyl)phenoxy]-N,N-dimethylacetamide 29 (100 mg, 60% yield)as a white solid. ¹H NMR (300 MHz, CD₃OD) δ 1.55 (m, 4H), 2.55 (m, 2H),2.66 (m, 2H), 2.90 (s, 3H), 3.05 (s, 3H), 4.70 (s, 2H), 6.80 (d, 2H),7.05 (d, 2H). m/z (ESI): 251 [C₁₄H₂₂N₂O₂+H]³⁰ .

2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-N,N-dimethylacetamidehydrochloride (30, PSA 16313)

A solution of 2-[4-(4-aminobutyl)phenoxy]-N,N-dimethylacetamide 29 (67mg, 0.27 mmol) in absolute ethanol (1 mL) was stirred at 65° C. for 30min, after which1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (110 mg, 0.29 mmol) was added in one portion. The reactionmixture was stirred at that temperature for 3 h and then cooled to roomtemperature. The reaction mixture was concentrated by rotaryevaporation. The crude residue was triturated with water and filtered.The filter cake was purified by flash silica gel column chromatographyeluting with dichloromethane/methanol/concentrated ammonium hydroxide(200:10:0, 200:10:1, v/v) to give2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}-phenoxy)-N,N-dimethylacetamideas a yellow solid (35 mg, 28% yield). This solid was dissolved inmethanol (2 mL) and added to 4 N aqueous HCl (4 drops). Concentration invacuo gave2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenoxy)-N,N-dimethylacetamidehydrochloride (30, PSA 16313). mp 130-132° C. (decomposed). ¹H NMR (300MHz, CD₃OD) δ 1.69 (m, 4H), 2.60 (m, 2H), 2.95 (s, 3H), 3.10 (s, 3H),3.35 (m, 2H), 4.75 (s, 2H), 6.80 (d, 2H), 7.10 (d, 2H). m/z (ESI): 463[C₂₀H₂₇ClN₈O₃+H]⁺.

Example 8 Synthesis of2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]-butyl}phenoxy)-N-(1H-imidazol-2-yl)acetamidedihydrochloride (PSA 16437)

[4-(4-tert-Butoxycarbonylaminobutyl)phenoxy]acetic acid methyl ester(32)

A mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid tert-butyl ester 31(1.00 g, 3.78 mmol), potassium carbonate (0.627 g, 4.54 mmol), sodiumiodide (0.567 g, 3.78 mmol), and methyl bromoacetate (0.40 mL, 4.21mmol) in anhydrous DMF (8 mL) was stirred at room temperature for 14 h.The reaction mixture was then diluted with ethyl acetate (100 mL) andhexanes (20 mL), washed with water (20 mL×4) and brine (30 mL), andconcentrated under reduced pressure to afford the desired product 32 asa yellow oil (1.28 g, 100% yield) which was used for the next stepwithout further purification. ¹H NMR (300 MHz, CDCl₃) δ 1.40 (s, 9H),1.41-1.65 (m, 4H), 2.49-2.60 (m, 2H), 3.02-3.16 (m, 2H), 3.79 (s, 3H),4.45 (br s, 1H), 4.59 (s, 2H), 6.79 (d, 2H), 7.05 (d, 2H). m/z (ESI):338 [C₁₈H₂₇NO₅+H]⁺.

[4-(4-tert-Butoxycarbonylaminobutyl)phenoxy]acetic acid (33)

A solution of [4-(4-tert-butoxycarbonylaminobutyl)phenoxy]acetic acidmethyl ester 32 (1.28 g, 3.78 mmol) in methanol (80 mL) was added withcrushed potassium hydroxide (2.50 g, 85%, 37.8 mmol) and the mixture wasstirred at room temperature for 5 h. Solvent was removed by rotaryevaporation. The residue was taken up in water and acidified to pH ˜1with 6N aqueous HCl, and extracted with dichloromethane. The combinedorganics were washed with brine, dried over Na₂SO₄, and concentrated tocomplete dryness to afford the desired product 33 as a white solid (1.19g, 97% yield). ¹H NMR (300 MHz, CD₃OD) δ 1.41 (s, 9H), 1.42-1.70 (m,4H), 2.45-2.60 (m, 2H), 3.00-3.20 (m, 2H), 4.60 (s, 2H), 6.80 (d, 2H),7.08 (d, 2H). m/z (ESI): 322 [C₁₇H₂₅NO₅—H]⁻.

(4-{4-[(1H-Imidazol-2-yl-carbamoyl)methoxy]phenyl}butyl)carbamic acidtert-butyl ester (34)

[4-(4-tert-Butoxycarbonylaminobutyl)phenoxy]acetic acid 33 (1.19 g, 3.68mmol) was dissolved in anhydrous THF (10 mL), CH₂Cl₂ (10 mL) and CH₃CN(5 mL). To the solution were sequentially added HOAt (200 mg, 1.47mmol), DMAP (135 mg, 1.10 mmol), and diisopropylethylamine (3.2 mL,18.40 mmol), followed by the addition of EDC.HCl (1.03 g, 5.35 mmol).The reaction mixture was stirred at room temperature for 15 min. Aminoimidazole sulfate (583 mg, 4.41 mmol) was then added and stirring wascontinued for 48 h. Solvents were removed by rotary evaporation. Theresidue was taken up in CH₂Cl₂ (250 mL), washed with water and brine,and concentrated under reduced pressure. Flash silica gel columnchromatography eluting with methanol/dichloromethane (1:30, 1:20, v/v)gave the desired amide as a white solid (0.95 g, 66% yield). ¹H NMR (300MHz, CD₃OD) δ 1.40 (s, 9H), 1.42-1.70 (m, 4H), 2.48-2.60 (m, 2H),3.00-3.20 (m, 2H), 4.65 (s, 2H), 6.79-6.89 (m, 4H), 7.10 (d, 2H). m/z(ESI): 389 [C₂₀H₂₈N₄O₄ ⁺H]⁺.

2-[4-(4-Aminobutyl)phenoxy]-N-(1H-imidazol-2-yl)acetamidedihydrochloride (35)

(4-{4-[(1H-Imidazol-2-yl-carbamoyl)methoxy]phenyl}butyl)carbamic acidtert-butyl ester 34 (950 mg, 2.45 mmol) was treated with HCl (4 M indioxane, 24 mL, 96 mmol) at room temperature for 12 h. The reactionmixture was concentrated in vacuo and further co-evaporated withdichloromethane and methanol, and dried under high vacuum. The desiredproduct was obtained as a white solid (779 mg, 98%) and used directlywithout further purification. ¹H NMR (300 MHz, CD₃OD) δ 1.59-1.74 (m,4H), 2.55-2.67 (m, 2H), 2.85-2.98 (m, 2H), 4.80 (s, 2H), 7.00 (d, 2H),7.18 (d, 2H), 7.19 (s, 2H). m/z (ESI): 289 [C₁₅H₂₀N₄O₂+H]⁺.

2-(4-{4-[N′-(3,5-Diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-N-(1H-imidazol-2-yl)acetamidedihydrochloride (36, PSA 16437)

A solution of 2-[4-(4-aminobutyl)phenoxy]-N-(1H-imidazol-2-yl)acetamidedihydrochloride 35 (99 mg, 0.27 mmol) and diisopropylethylamine (0.27mL, 1.53 mmol) in absolute ethanol (4 mL) and anhydrous methanol (3 mL)was stirred at 70° C. for 30 min, after which1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (130 mg, 0.34 mmol) was added in one portion. The reactionmixture was stirred for 3 h and then cooled to room temperature. Theyellow insolubles were removed by suction filtration and the liquidfiltrate was concentrated by rotary evaporation. The crude residue waspurified by flash silica gel column chromatography eluting withdichloromethane/methanol/concentrated ammonium hydroxide (200:10:0,200:10:1, 150:10:1, and 100:10:1, v/v) to give2-(4-{4-[NA-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)-N-(1H-imidazol-2-yl)-acetamideas a yellow solid (44 mg, 29% yield). The free base thus obtained wasdissolved in methanol and treated with 4 drops of 4 N aqueous HCl. Thesolution was concentrated under reduced pressure and further dried undervacuum to give the final compound 36. mp 172-174° C. ¹H NMR (300 MHz,CD₃OD) δ 1.61-1.77 (m, 4H), 2.58-2.70 (m, 2H), 3.32-3.40 (m, 2H), 4.80(s, 2H), 7.00 (d, 2H), 7.18 (d, 2H), 7.20 (s, 2H). m/z (ESI): 501[C₂₁H₂₅ClN₁₀O₃+H]⁺.

Example 9 Synthesis ofN-carbamoylmethyl-2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino]butyl}phenoxy)acetamide(PSA 16314)

(4-{4-[(Carbamoylmethylcarbamoyl)methoxy]phenyl}butyl)carbamic acidbenzyl ester (37)

Compound 1 (0.50 g, 1.77 mmol) was dissolved in DMF (10 mL). To thesolution was added crushed NaOH (0.107 g, 2.66 mmol). The mixture wasstirred at room temperature for 30 min. 2-Bromoacetamide (0.367 g, 2.66mmol) was added. The reaction was further stirred at room temperatureovernight, quenched with water (2 mL) and partitioned between water anddichloromethane (each 50 mL). The organic layer was separated, washedwith water (2×50 mL), dried over anhydrous Na₂SO₄ and concentrated undervacuum. The residue was purified on silica gel, eluting with a mixtureof methanol/dichloromethane (7:93, v/v), to afford the desired product37 (0.131 g, 18% yield) as a white solids. ¹H NMR (300 MHz, CDCl₃): δ1.58 (m, 4H), 2.60 (t, 2H), 3.20 (m, 2H), 4.04 (d, 2H), 4.54 (s, 2H),4.75 (br, 2H), 5.12 (s, 2H), 5.43 (br, 1H), 5.80 (br, 1H), 6.85 (d, 2H),7.12 (d, 2H), 7.36 (m, 5H). m/z (APCI): 414 [C₂₂H₂₇N₃O₅+H]⁺.

2-[4-(4-Aminobutyl)phenoxy]-N-carbamoylmethylacetamide (38)

Compound 37 (130 mg, 0.314 mmol) was dissolved in EtOH and THF (14 mL,1/1 ratio). The reaction vessel was purged with nitrogen both before andafter the catalyst (100 mg, 10% Pd/C, 50% wet) was added. The mixturewas stirred under hydrogen atmosphere (1 atm) overnight. After purgingwith nitrogen, the catalyst was vacuum filtered and washed with ethanol(3×5 mL). The combined filtrates were concentrated under vacuum. Theresidue was chromatographed on silica gel, eluting with a mixture ofconcentrated ammonium hydroxide/methanol/dichloromethane (2:20:88, v/v),to afford the desired product 38 (80 mg, 91% yield) as a white solid. ¹HNMR (300 MHz, CD₃OD): δ 1.62 (m, 4H), 2.60 (t, 2H), 2.75 (t, 2H), 3.92(s, 2H), 4.54 (s, 2H), 6.92 (d, 2H), 7.14 (d, 2H).

N-Carbamoylmethyl-2-(4-{4-[N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)-guanidino]butyl}phenoxy)acetamide(39, PSA 1634)

Compound 38 (79 mg, 0.283 mmol) was dissolved in a mixture of absoluteethanol (5 mL) and Hunig's base (0.25 mL, 1.41 mmol) at 65° C. over 10min. To the solution was added1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (132 mg, 0.34 mmol) in one portion. The newly resultingreaction mixture was continuously stirred for an additional 2 h beforeit was cooled down to ambient temperature and subsequently concentratedunder vacuum. The resulting residue was purified by chromatographyeluting with methanol/dichloromethane/concentrated ammonium hydroxide(Oct. 2, 1988, v/v) to afford the free base (93 mg, 67% yield) as ayellow solid. The HCl salt was made using the following procedure: 45 mgof the free base was suspended in ethanol (2 mL) and treated withconcentrated HCl (12 N, 0.5 mL) for 10 min. All liquid was thencompletely removed under vacuum to afford 39 (47 mg). mp 178-180° C.(decomposed). ¹H NMR (300 MHz, DMSO-d₆): δ 1.61 (m, 4H), 2.58 (t, 2H),3.32 (m, 2H), 3.70 (s, 2H), 4.48 (s, 2H), 6.93 (d, 2H), 7.08 (br, 1H),7.13 (d, 2H), 7.36 (br, 1H), 7.44 (br, 2H), 8.17 (t, 1H), 8.74 (br, 1H),8.90 (br, 2H), 9.18 (t, 1H), 10.48 (br, 1H). m/z (APCI): 492[C₂₀H₂₆ClN₉O₄+H]⁺.

Example 10 Synthesis ofN-[4-(4-cyanomethoxyphenyl)butyl]-N′-(3,5-diamino-6-chloro-pyrazine-2-carbonyl)guanidine(PSA 16208)

[4-(4-Cyanomethoxyphenyl)butyl]carbamic acid tert-butyl ester (40)

A mixture of [4-(4-hydroxyphenyl)butyl]carbamic acid tert-butyl ester 31(0.365 g, 1.37 mmol) and Cs₂CO₃ (0.672 g, 2.06 mmol) in anhydrous DMF (8mL) was heated at 65° C. for 30 min. Iodoacetonitrile (0.276 g, 1.651mmol) was then added to the mixture in one portion. The mixture wasstirred at 65° C. ovenight, and then cooled to room temperature. Theprecipitated solid was filtered, and the filtrate was partitionedbetween water and dichloromethane (each 50 mL). The organic layer wasseparated, washed with brine (3×50 mL), dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The residue was chromatographed on silicagel, eluting with a mixture of diethyl ether/dichloromethane (6:94,v/v), to afford the desired product 40 (0.109 g, 38% yield) as acolorless viscous oil. ¹H NMR (300 MHz, CDCl₃): δ 1.43 (s, 9H), 1.57 (m,4H), 2.60 (t, 2H), 3.15 (m, 2H), 4.49 (br, 1H), 4.75 (s, 2H), 6.91 (d,2H), 7.13 (d, 2H).

[4-(4-Aminobutyl)phenoxy]acetonitrile (41)

Compound 40 (0.105 g, 0.345 mmol) was dissolved in dichloromethane (10mL). Trifluoroacetic acid (2 mL) was added in one portion. The mixturewas stirred at room temperature for 2 h, and then concentrated undervacuum to dryness. The crude residue was used directly without furtherpurification. ¹H NMR (300 MHz, CD₃OD): δ 1.60-1.75 (m, 4H), 2.65 (t,2H), 2.92 (t, 2H), 4.38 (s, 2H), 6.96 (d, 2H), 7.20 (d, 2H). m/z (APCI):205 [C₁₂H₁₆N₂O+H]⁺.

N-[4-(4-Cyanomethoxyphenyl)butyl]-N′-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidine(42, PSA 16208)

A mixture of compound 41 (0.070 g, 0.345 mmol) and Hunig's base (0.3 mL,1.72 mmol) in anhydrous ethanol was heated at 65° C. for 20 min. To thesolution was added1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (0.148 g, 0.379 mmol) in one portion. The heating wascontinued for another 2 h. The reaction mixture was then concentratedunder vacuum. The residue was chromatographed by flash columnchromatography and further purified by preparative TLC, eluting withmethanol/dichloromethane/concentrated ammonium hydroxide (Oct. 1, 1989,v/v), to afford the desired product 42 (0.031 g, 22%) as a yellow solid.mp 129-132° C. ¹H NMR (300 MHz, CD₃OD): δ 1.72 (m, 4H), 2.68 (t, 2H),3.32 (m, 2H), 4.92 (s, 2H), 6.95 (d, 2H), 7.22 (d, 2H); m/z (APCI): 417[C₁₈H₂₁ClN₈O₂+H]⁺.

Example 11 Synthesis ofN-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-(4-{4-[3-(2,3-dihydroxypropoxy)-2-hydroxypropoxy]phenyl}butyl)guanidine(PSA 15143)

(4-{4-[3-(2,3-Dihydroxypropoxy)-2-hydroxypropoxy]phenyl}butyl)carbamicacid benzyl ester (43).

A solution containing compound 1 (2.0 g, 6.68 mmol), triethylamine(0.093 mL, 0.668 mmol) and anhydrous ethanol (2.2 mL) was heated at 70°C. for 1 h. Oxiranylmethanol (0.5 mL, 6.68 mmol) was added every hourfor a total of 4 h (the total amount of oxiranylmethanol added was 2.0ml, 26.72 mmol). The reaction was concentrated under vacuum. The residuewas chromatographed on silica gel with the elution of a mixture ofmethanol/dichloromethane (3:97, v/v) to provide 168 mg (4.6% yield) ofthe desired product 43. m/z (APCI): 448 [C₂₄H₃₃NO₇+H]⁺.

3-{3-[4-(4-Aminobutylphenoxy]-2-hydroxypropoxy}propane-1,2-diol (44)

A solution containing the compound 43 (0.15 g, 0.34 mmol) in ethanol(1.5 mL) was purged with nitrogen before and after the catalyst (0.15 g,10% Pd/C, 50% wet) was added. The reaction mixture was placed underhydrogenation atmosphere for 45 min. The catalyst was vacuum filteredthrough diatomaceous earth and washed with ethanol (3×2 mL). Thecombined filtrates were concentrated under vacuum. The residue waschromatographed on silica gel, eluting withmethanol/dichloromethane/concentrated ammonium (25/2.5/73.5, v/v), toafford the desired product 44 (0.053 g, 51% yield) as a colorless,viscous oil. ¹H NMR (300 MHz, CD₃OD): δ 1.52 (m, 4H), 2.55 (t, 2H), 2.65(t, 2H), 3.61 (m, 10H), 6.85 (d, 2H), 7.09 (d, 2H). m/z (APCI): 314[C₁₆H₂₇NO₅+H]⁺.

N-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-(4-{4-[3-(2,3-dihydroxypropoxy)-2-hydroxypropoxy]phenyl}butyl)guanidine(45, PSA 15143)

Compound 44 (50 mg, 0.159 mmol) was dissolved in a mixture of absoluteethanol (0.5 mL) and triethylamine (0.076 mL, 0.541 mmol) at 65° C. over15 min. To the solution was added1-(3,5-diamino-6-chloropyrazine-2-carbonyl)-2-methylisothioureahydriodide (74 mg, 0.191 mmol). The reaction mixture was stirred at theabove temperature for an additional 50 min, cooled down to ambienttemperature and subsequently concentrated under vacuum. The residue waschromatographed on silica gel, eluting withmethanol/dichloromethane/concentrated ammonium hydroxide (Oct. 1, 1940,v/v) to afford the desired product 45 (53 mg, 36% yield) as a yellowsolid. mp 73-82° C. (decomposed). ¹H NMR (300 MHz, CD₃OD): δ 1.70 (m,4H), 2.55 (m, 2H), 3.22 (m, 2H), 3.65 (m, 7H), 3.98 (m, 3H), 6.86 (d,2H), 7.08 (d, 2H). m/z (APCI): 526 [C₂₂H₃₂ClN₇O₆+H]⁺.

Example 12

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 300 MHz¹H NMR Spectrum (DMSO-d₆) Consistent Melting Point 108-110° C. dec HPLCAnalysis 96.5% (area percent), Polarity dC18 Column, Detector @ 220 nmMiscellaneous Tests: ESI Mass Spectrum m/z 527 [C₂₁H₃₁ClN₈O₄S + H]⁺

Example 13

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 300 MHz¹H NMR Spectrum (DMSO-d₆) Consistent Melting Point 153-155° C. dec HPLCAnalysis 96.3% (area percent), Polarity dC18 Column, Detector @ 220 nmMiscellaneous Tests: ESI Mass Spectrum m/z 465 [C₁₉H₂₅ClN₈O₂S + H]⁺

Example 14

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 500 MHz¹H NMR Spectrum (CD₃OD) Consistent Melting Point 115-116° C. HPLCAnalysis 97.1% (area percent), Polarity dC18 Column, Detector @ 220 nmMiscellaneous Tests: ESI Mass Spectrum m/z 639 [C₃₀H₃₅ClN₈O₆ + H]⁺

Example 15

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 300 MHz¹H NMR Spectrum (CD₃OD) Consistent Melting Point 190-192° C. HPLCAnalysis 97.9% (area percent), Polarity dC18 Column, Detector @ 220 nmMiscellaneous Tests: ESI Mass Spectrum m/z 476 [C₂₀H₂₆ClN₉O₃ + H]⁺

Example 16

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 300 MHz¹H NMR Spectrum (CD₃OD) Consistent Melting Point 124-126° C. dec HPLCAnalysis 95.2% (area percent), Polarity dC18 Column, Detector @ 220 nmMiscellaneous Tests: ESI Mass Spectrum m/z 441 [C₁₆H₂₁ClN₈O₃S + H]⁺

Example 17

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 500 MHz¹H NMR Spectrum (CD₃OD) Consistent Melting Point 189° C. dec HPLCAnalysis 95.0% (area percent), Polarity dC18 Column, Detector @ 220 nmMiscellaneous Tests: ESI Mass Spectrum m/z 503 [C₂₁H₂₇ClN₁₀O₃ + H]⁺

Example 18

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Pale yellow solid Identification: 300MHz ¹H NMR Spectrum (CD₃OD) Consistent Melting Point 195-197° C. HPLCAnalysis 97.4% (area percent), Polarity dC18 Column, Detector @ 220 nmMiscellaneous Tests: ESI Mass Spectrum m/z 477 [C₂₀H₂₅ClN₈O₄ + H]⁺

Example 19

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 300 MHz¹H NMR Spectrum (CD₃OD) Consistent Melting Point 210-212° C. dec HPLCAnalysis 95.5% (area percent), Polarity dC18 Column, Detector @ 220 nmMiscellaneous Tests: APCI Mass Spectrum m/z 486 [C₂₂H₂₈ClN₉O₂ + H]⁺

Example 20

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 300 MHz¹H NMR Spectrum (CD₃OD) Consistent Optical Rotation [α]²⁵ _(D) −7.8° (c0.46, Methanol) Melting Point 178-180° C. HPLC Analysis 97.0% (areapercent), Polarity dC18 Column, Detector @ 220 nm Miscellaneous Tests:ESI Mass Spectrum m/z 490 [C₂₁H₂₈ClN₉O₃ + H]⁺

Example 21

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 300 MHz¹H NMR Spectrum (CD₃OD) Consistent Optical Rotation [α]²⁵ _(D) +0.5° (c0.35, Methanol) Melting Point 215° C. dec HPLC Analysis 96.1% (areapercent), Polarity dC18 Column, Detector @ 220 nm Miscellaneous Tests:ESI Mass Spectrum m/z 462 [C₂₀H₂₈ClN₉O₂ + H]⁺

Example 22

Utilizing the procedures set forth above, the following cappedpyrazinoylguanidine was prepared.

TEST RESULT/REFERENCE Description Yellow solid Identification: 300 MHz¹H NMR Spectrum (CD₃OD) Consistent Optical Rotation [α]²⁵ _(D) +4.1° (c0.30, Methanol) Melting Point 230° C. dec HPLC Analysis 95.3% (areapercent), Polarity dC18 Column, Detector @ 220 nm Miscellaneous Tests:ESI Mass Spectrum m/z 463 [C₂₀H₂₇ClN₈O₃ + H]⁺

Example 23

Sodium Channel Blocking Activity of Selected CappedPyrazinoylguanidines.

PSA EC₅₀(nM) Fold Amiloride** (PSA 4022 = 100) 15143 7 ± 3 (n = 3) 107 ±11 (n = 3) 16208 11 ± 4 (n = 6) 52 ± 21 (n = 6) 16314 13 ± 2 (n = 4) 41± 6 (n = 4) 16313 15 ± 4 (n = 4) 41 ± 7 (n = 4) 16437 13 ± 7 (n = 7) 77± 53 (n = 7) 17482 16 ± 4 (n = 3) 39 ± 6 (n = 3) 17846 11 ± 6 (n = 4)104 ± 49 (n = 4) 17926 25 ± 9 (n = 6) 29 ± 12 (n = 6) 17927 13 ± 4 (n =3) 83 ± 26 (n = 3) 18211 10 ± 4 (n = 3) 112 ± 52 (n = 2) 18212 27 ± 17(n = 4) 32 ± 16 (n = 4) 18229 15 ± 6 (n = 3) 49 ± 15 (n = 3) 18361 11 ±4 (n = 3) 76 ± 25 (n = 3) 18592 8 ± 4 (n = 2) 136 ± 58 (n = 2) 18593 48± 16 (n = 6) 13 ± 4 (n = 4) 19007 18 ± 13 (n = 4) 42 ± 17 (n = 4) 190089 ± 1 (n = 4) 54 ± 6 (n = 4) 19912 26 ± 8 (n = 4) 32 ± 10 (n = 4) 2302212 ± 3 (n = 4) 79 ± 15 (n = 4) 24406 8 ± 3 (n = 6) 107 ± 38 (n = 6)24407 32 ± 11 (n = 10) 23 ± 4 (n = 10) 24851 28 ± 13 (n = 8) 25 ± 10 (n= 8) **Relative potency for PSA 4022 = 100 using EC₅₀ from PSA 4022 insame run

Methods

Animal Preparation Adult ewes (ranging in weight from 25 to 35 kg) wererestrained in an upright position in a specialized body harness adaptedto a modified shopping cart. The animals' heads were immobilized andlocal anesthesia of the nasal passage was induced with 2% lidocaine. Theanimals were then nasally intubated with a 7.5 mm internal diameterendotracheal tube (ETT). The cuff of the ETT was placed just below thevocal cords and its position was verified with a flexible bronchoscope.After intubation the animals were allowed to equilibrate forapproximately 20 minutes prior to initiating measurements of mucociliaryclearance.

Administration of Radio-aerosol: Aerosols of ^(99m)Tc-Human serumalbumin (3.1 mg/ml; containing approximately 20 mCi) were generatedusing a Raindrop Nebulizer which produces a droplet with a medianaerodynamic diameter of 3.6 μm. The nebulizer was connected to adosimetry system consisting of a solenoid valve and a source ofcompressed air (20 psi). The output of the nebulizer was directed into aplastic T connector; one end of which was connected to the endotrachealtube, the other was connected to a piston respirator. The system wasactivated for one second at the onset of the respirator's inspiratorycycle. The respirator was set at a tidal volume of 500 mL, aninspiratory to expiratory ratio of 1:1, and at a rate of 20 breaths perminute to maximize the central airway deposition. The sheep breathed theradio-labeled aerosol for 5 minutes. A gamma camera was used to measurethe clearance of ^(99m)Tc-Human serum albumin from the airways. Thecamera was positioned above the animal's back with the sheep in anatural upright position supported in a cart so that the field of imagewas perpendicular to the animal's spinal cord. External radio-labeledmarkers were placed on the sheep to ensure proper alignment under thegamma camera. All images were stored in a computer integrated with thegamma camera. A region of interest was traced over the imagecorresponding to the right lung of the sheep and the counts wererecorded. The counts were corrected for decay and expressed aspercentage of radioactivity present in the initial baseline image. Theleft lung was excluded from the analysis because its outlines aresuperimposed over the stomach and counts can be swallowed and enter thestomach as radio-labeled mucus.

Treatment Protocol (Assessment of activity at t-zero): A baselinedeposition image was obtained immediately after radio-aerosoladministration. At time zero, after acquisition of the baseline image,vehicle control (distilled water), positive control (amiloride), orexperimental compounds were aerosolized from a 4 ml volume using a PariLC JetPlus nebulizer to free-breathing animals. The nebulizer was drivenby compressed air with a flow of 8 liters per minute. The time todeliver the solution was 10 to 12 minutes. Animals were extubatedimmediately following delivery of the total dose in order to preventfalse elevations in counts caused by aspiration of excess radio-tracerfrom the ETT. Serial images of the lung were obtained at 15-minuteintervals during the first 2 hours after dosing and hourly for the next6 hours after dosing for a total observation period of 8 hours. Awashout period of at least 7 days separated dosing sessions withdifferent experimental agents. Treatment Protocol (Assessment ofActivity at t−4 hours): The following variation of the standard protocolwas used to assess the durability of response following a singleexposure to vehicle control (distilled water), positive controlcompounds (amiloride or benzamil), or investigational agents. At timezero, vehicle control (distilled water), positive control (amiloride),or investigational compounds were aerosolized from a 4 ml volume using aPari LC JetPlus nebulizer to free-breathing animals. The nebulizer wasdriven by compressed air with a flow of 8 liters per minute. The time todeliver the solution was 10 to 12 minutes. Animals were restrained in anupright position in a specialized body harness for 4 hours. At the endof the 4-hour period animals received a single dose of aerosolized^(99m)Tc-Human serum albumin (3.1 mg/ml; containing approximately 20mCi) from a Raindrop Nebulizer. Animals were extubated immediatelyfollowing delivery of the total dose of radio-tracer. A baselinedeposition image was obtained immediately after radio-aerosoladministration. Serial images of the lung were obtained at 15-minuteintervals during the first 2 hours after administration of theradio-tracer (representing hours 4 through 6 after drug administration)and hourly for the next 2 hours after dosing for a total observationperiod of 4 hours. A washout period of at least 7 days separated dosingsessions with different experimental agents.

Statistics: Data were analyzed using SYSTAT for Windows, version 5. Datawere analyzed using a two-way repeated ANOVA (to assess overalleffects), followed by a paried t-test to identify differences betweenspecific pairs. Significance was accepted when P was less than or equalto 0.05. Slope values (calculated from data collected during the initial45 minutes after dosing in the t-zero assessment) for mean MCC curveswere calculated using linear least square regression to assessdifferences in the initial rates during the rapid clearance phase.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A compound represented by formula (I):

wherein X is hydrogen, halogen, trifluoromethyl, lower alkyl,unsubstituted or substituted phenyl, lower alkyl-thio, phenyl-loweralkyl-thio, lower alkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl; Y ishydrogen, hydroxyl, mercapto, lower alkoxy, lower alkyl-thio, halogen,lower alkyl, unsubstituted or substituted mononuclear aryl, or —N(R²)₂;R¹ is hydrogen or lower alkyl; each R² is, independently, —R⁷,—(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-Z_(g)-R⁷, —(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—(CH₂)_(n), —CO₂R⁷, or

R³ and R⁴ are each, independently, hydrogen, a group represented byformula (A), lower alkyl, hydroxy lower alkyl, phenyl, phenyl-loweralkyl, (halophenyl)-lower alkyl, lower-(alkylphenylalkyl), lower(alkoxyphenyl)-lower alkyl, naphthyl-lower alkyl, or pyridyl-loweralkyl, with the proviso that at least one of R³ and R⁴ is a grouprepresented by formula (A):

wherein each R^(L) is, independently, —R⁷, —(CH₂)_(n)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂), —NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(n)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)-(Z)_(g)-R⁷,—O—(CH₂)_(m)-(Z)_(g)-R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR¹,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(n), —CO₂R⁷—OSO₃H, —O-glucuronide, —O-glucose,

each o is, independently, an integer from 0 to 10; each p is an integerfrom 0 to 10; with the proviso that the sum of o and p in eachcontiguous chain is from 1 to 10; each x is, independently, O, NR¹⁰,C(═O), CHOH, C(═N—R¹⁰), CHNR⁷R¹⁰, or represents a single bond; whereineach R⁵ is, independently, Link-(CH₂)_(n)-CAP,Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, Link-(CH₂CH₂O)_(n)—CH₂—CAP,Link-(CH₂CH₂O)_(m)—CH₂CH₂-CAP, Link-(CH₂)_(n)-(Z)_(g)-CAP,Link-(CH₂)_(n)(Z)_(g)-(CH₂)_(m)-CAP,Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)—CAP,Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³-(Z)_(g)-CAP,Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³-(Z)_(g)-CAP,Link-(CH₂)_(m)-(Z)_(g)-(CH₂)_(m)-CAP, Link-NH—C(═O)—NH—(CH₂)_(m)—CAP,Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)—C(═O)NR¹⁰R¹⁰, Link-(CH₂)_(m),—C(═O)NR¹³—(CH₂)_(m)—CAP, Link-(CH₂)_(m)—C(═O)NR¹¹R¹¹,Link-(CH₂)_(m)—C(═O)NR¹²R¹²,Link-(CH₂)_(n)-(Z)_(g)-(CH₂)_(m)-(Z)_(g)-CAP,Link-Z_(g)-(CH₂)_(m)-Het-(CH₂)_(m)—CAP; each Link is, independently,—O—, —(CH₂)_(m)—, —O(CH₂)_(m)—, —NR¹³—C(═O)—NR³, —NR¹³—C(═O)—(CH₂)_(m)—,—C(═O)NR¹³—(CH₂)_(m), —(CH₂)_(n)-Z_(g)-(CH₂)_(n), —S—, —SO—, —SO₂—,—SO₂NR⁷—, —SO₂NR¹⁰—, or -Het-; each CAP is, independently,thiazolidinedione, oxazolidinedione, heteroaryl-C(═O)N R¹³R¹³,heteroaryl-W, —CN, —O—C(═S)NR¹³R¹³, -Z_(g)R¹³,—CR¹⁰(Z_(g)R¹³)(Z_(g)R¹³), —C(═O)OAr, —C(═O)NR¹³Ar, imidazoline,tetrazole, tetrazole amide, —SO₂NHR¹³, —SO₂NH—C(R¹³R¹³)-(Z)_(g)-R¹³, acyclic sugar or oligosaccharide, a cyclic amino sugar oroligosaccharide,

each Ar is, independently, phenyl, substituted phenyl, wherein thesubstituents of the substituted phenyl are 1-3 substituentsindependently selected from the group consisting of OH, OCH₃, NR¹³R¹³,Cl, F, and CH₃, or heteroaryl; each W is independently,thiazolidinedione, oxazolidinedione, heteroaryl-C(═O)N R¹³R¹³, —CN,—O—C(═S)NR¹³R¹³, -Z_(g)R¹³, —CR¹⁰(ZgR¹³)(ZgR¹³), —C(═O)OAr,—C(═O)NR¹³Ar, imidazoline, tetrazole, tetrazole amide, —SO₂NHR¹³,—SO₂NH—C(R¹³R¹³)-(Z)_(g)-R¹³, a cyclic sugar or oligosaccharide, acyclic amino sugar or oligosaccharide,

each R⁶ is, independently, —R⁷, —OR⁷, —OR¹¹, —N(R⁷)₂, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(m)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, O—(CH₂CH₂O),—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰, —O—(CH₂)_(m)—C(═O)NR⁷R¹⁰,—(CH₂)_(n)-(Z)_(g)-R⁷, —O—(CH₂)_(m)-(Z)_(g)R⁷,—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂), —CO₂R⁷,O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

wherein when two R⁶ are —OR¹¹ and are located adjacent to each other ona phenyl ring, the alkyl moieties of the two R⁶ may be bonded togetherto form a methylenedioxy group; each R⁷ is, independently, hydrogenlower alkyl, phenyl, substituted phenyl or —CH₂(CHOR)⁸ _(m)—R¹⁰; each R⁸is, independently, hydrogen, lower alkyl, —C(═O)—R¹¹, glucuronide,2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R¹³, —CON(R¹³)₂, —SO₂CH₂R¹³, or—C(═O)R¹³; each R¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R¹³,—C(═O)NR³R¹³, —C(═O)R¹³, or —(CH₂)_(m)—(CHOH)_(m)—CH₂OH; each Z is,independently, CHOH, C(═O), —(CH₂)_(n)—, CHNR³R³, C═NR³, or NR¹³; eachR¹¹ is, independently, lower alkyl; each R¹² is independently, —SO₂CH₃,—CO₂R¹³, —C(═O)NR¹³R¹³, —C(═O)R¹³, or —CH₂—(CHOH)_(n)—CH₂OH; each R¹³is, independently, hydrogen, R⁷, R¹⁰,

with the proviso that NR¹³R¹³ can be joined on itself to form a ringcomprising one of the following:

each Het is independently, —NR_(—3), —S—, —SO—, or —SO₂—; —O—,—SO₂NR¹³—, —NHSO₂—, —NR¹³CO—, or —CONR¹³; each g is, independently, aninteger from 1 to 6; each m is, independently, an integer from 1 to 7;each n is, independently, an integer from 0 to 7; each Q is,independently, C—R⁵, C—R⁶, or a nitrogen atom, wherein at most three Qin a ring are nitrogen atoms; each V is, independently,

with the proviso that when V is attached directly to a nitrogen atom,then V can also be, independently, R⁷, R¹⁰, or (R¹¹)₂; with the provisothat, when any two —CH₂OR⁸ groups are located 1, 2 or 1,3-with respectto each other, the R⁹ groups may be joined to form a cyclic mono- ordi-substituted 1,3-dioxane or 1,3-dioxolane; or a pharmaceuticallyacceptable salt thereof; and inclusive of all enantiomers,diastereomers, and racemic mixtures thereof. 2-114. (canceled)